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Stages of the Cellular Response to Stress and Injurious Stimuli CELLULAR ADAPTATION
DAVAO DOCTORS COLLEGE
- Changes made by a cell in response to stress or stimuli
MEDICAL LABORATORY SCIENCE DEPARTMENT - May be physiologic or pathologic
STUDENT NOTES: GPHCT Forms of Adaptation
1. Hypertrophy
Increased Cell Size  Increased Organ Size
REVIEW OF BASIC HISTOLOGY INTRODUCTION TO GENERAL PATHOLOGY Due to increased protein synthesis
Most common stimulus: Increased Workload
Histology Study of tissues and their structure. Pathology (pathos: suffering; logos: study)
Examples:
Germ Layers Group of cells that form during embryonic - study of diseases at cellular, tissue and organ level  bulging muscles of bodybuilders,
A. Ectoderm development. The layers will eventually - bridge between mediZcine and science; it is the scientific  estrogen-induced enlargement of uterus during pregnancy
B. Mesoderm differentiate into various tissues and organs. foundation of medicine
C. Endoderm Remember: *body builders win trophy*
Divisions of Pathology 2. Hyperplasia
Types of Tissues
A. Epithelial Covering epithelia: Lines surfaces A. Gross Pathology Macroscopic examination of tissues Increased Cell Number  Increased Organ Mass
Glandular epithelia: secrete substances and organs Due to proliferative actions of growth factor, and/or stem cells
B. Microscopic Pathology
B. Connective Most abundant; Binds, protects, supports Physiologic:
i. Anatomic Surgical
• 1. Normal cells have defined structure and can perform limited  Hormonal hyperplasia - Breast during puberty or
C. Muscular Highly specialized; Movement Pathology Biopsy (living), Autopsy (dead)
• functions based on their specialization, metabolism, and pregnancy
 Histopathology availability of metabolic substrates. They can handle physiologic
D. Nervous Sensation, integration, response ii. Clinical Pathology • Hematology demands through homeostasis.  Compensatory hyperplasia - Liver cells regeneration
• Microbiology Pathologic:
• Clinical Chemistry Homeostasis - act of maintaining a steady state
■ Excess Hormonal Stimulation
• Immunology/Serology
 Increased Estrogen  Endometrial hyperplasia 
• Clinical Microscopy 2. When there is a slightly severe stress, or some pathologic
• Parasitology
abnormal menstrual bleeding
Importance of Histology in General Pathology stimuli, cells undergo adaptation in order to survive and
■ Excess Growth Hormone Stimulation
Rudolf Virchow – father of modern pathology continue to function.
 Disease processes affect tissues in distinctive ways, which Papillomavirus mucosal lesions
depend on the type of tissue, and the disease itself. Disease Adaptation - reversible structural and functional response of 3. Atrophy
 These processes may cause them to die, to change their - Any change from a state of health as a result of certain cells to stress and stimuli
shape, to divide, to move or to invade other tissues. Any of Decreased Cell Size & Number  Reduce tissue/organ size
forms of stimuli and stress, which leads to impaired
these changes also affect the anatomy of the tissue. 3. But if the limits of adaptive response are exceeded, or when cells Due to decreased protein synthesis, and increased protein
physiological functioning degradation
 Understanding the changes that are characteristic of a are exposed to injurious stimuli (agents or stress), or deprived
disease requires a detailed knowledge of the normal histology of essential nutrients, cell injury occurs.
Four Aspects of a Disease Process Physiologic: as in puberty when the thymus and the lymphoid
of cells and tissues, and the range of normality. a. If the stimulus is mild and transient, the injury is
Etiology Cause or origin of the disease; might be organs decrease in size
genetic factors or acquired factors reversible. The cell may go back to its normal state.
 Knowing the type of tissue and their composition is important e.g. Atrophy of uterus after pregnancy
Mechanisms of the development of the b. If it is severe and progressive, the injury is irreversible.
in the selection of the appropriate histopathologic technique Pathogenesis
disease Cells that undergo irreversible injury will ultimately suffer Pathologic (Types):
and stain to be used.
cell death, which may be pathological or physiological. 1. Atrophy of disuse - decreased workload, thus
 These changes within cells and tissues can be visualized Sequence of events from initial stimulus to
ultimate expression of the disease diminished function of organ
using histopathologic techniques. Other types of stress can induce responses other than cellular e.g. skeletal muscle atrophy due to bedrest
How etiologic factors trigger cellular & adaptation, injury and death. The responses are the following:
2. Vascular - Diminished blood supply (ischemia)
molecular changes in a disease e.g. brain atrophy during atherosclerosis
A. Autophagy (self-eating) - starved cells eat its own components
Morphologic & Structural, biochemical and molecular during nutrient deprivation 3. Starvation - inadequate nutrition of cell
Molecular Changes alterations induced in the cells and organs of B. Intracellular accumulation of substances (such as proteins, e.g. muscle wasting (or cachexia) due to use of skeletal
the body as a result of the disease lipids, hyaline, glycogen, pigments) muscle as source of energy during protein malnutrition
Clinical Functional consequence of the changes C. Pathologic calcification – abnormal tissue deposition of calcum 4. Loss of endocrine stimulation - due to decrease of
Manifestations salts regulating hormones
Signs Effects that can be observed by others D. Cellular aging – progressive decline in the life span and e.g. breast atrophy after menopause due to loss of
Symptoms Effects apparent only to the patient functional capacity of cells estrogen stimulation
5. Pressure - as in growth of tumors, atrophy occurs when
Almost all forms of disease start with alterations in cells. Therefore, it tumors suppress the blood supply or by directly putting
is important to study the causes, mechanisms and morphologic and pressure to surrounding healthy cells
biochemical correlates of cell injury. 6. Exhaustion - due to increase in metabolism resulting to
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4. Metaplasia APOPTOSIS
Change in one cell type to another
INFLAMMATION
- Induced by a tightly regulated suicide program in which
Due to reprogramming of existing stem cells in normal tissue, - A protective universal response to tissue Classes of Inflammation
cells destined to die activate enzymes that degrade the
or of undifferentiated mesenchymal cells in order to damage (mechanical trauma, tissue 1. According to Duration
cells' own proteins and nuclear DNA
withstand adverse environment necrosis, infection)
- Presence of cleaved, active caspases (cysteine proteases i. Acute Sudden onset; usually mild and self-
- May be beneficial or harmful
that cleave aspartic acid residue) is a marker for cells limiting; Polymorphonuclear (PMNs)
Example: Functions:
undergoing apoptosis cells
> Habitual cigarette smokers - ciliated columnar cells of trachea 1. contain damage & isolate injury
- Cells break up into apoptotic bodies, which are tasty ii. Chronic Involves persistence of injurious
and bronchi are replaced by stratified squamous cells 2. destroy cause of injury (microorganism/toxins)
Barrett esophagus - squamous cells of esophagus are targets for phagocytes agent; often severe and progressive;
> 3. destroy resulting necrotic cells and tissues
replaced by intestinal-like columnar cells in response to Mononuclear cells
4. prepare tissue for healing & repair
refluxed acid 2. According to Character of Exudate
Reasons for Apoptosis in Following Conditions:
Harmful effects: i. Serous Out-pouring of relatively protein-poor
Physiologic Eliminates cells that are no longer needed, or those 1. Digestion of normal tissues fluid; common in cavities
that have served their purposes
2. Swelling
Pathologic Eliminates cells that are injured beyond repair ii. Fibrinous More severe injury 
3. Inappropriate inflammatory response
CELLULAR INJURY without eliciting host reaction more vascular permeability 
more protein leaking (such as
- Alteration in cell structure or function due to stress or Cardinal Signs:
fibrinogen which is the precursor of
pathologic stimuli NECROSIS 1. Rubor – redness
fibrin)
- Pathologic cell death 2. Calor – heat
iii. Catarrhal Increased blood flow to mucosal
Necrotic - tissue or organs with large numbers of dead cells 3. Tumor – swelling vessels, enlargement of secretory
Causes 4. Dolor – pain vessels  discharge of mucus and
Hypoxia Immunologic Reactions 5. Functio laesa – loss of function epithelial debris
Types of Necrosis According to Morphology
Physical Agents Genetic Abnormalities Components of Inflammation iv. Hemorrhagic Disruption of blood vessel wall 
Chemical Agents and Drugs Nutritional Imbalances 1. Coagulative Tissue is firm because architecture of dead tissue leakage of large number of RBCs
Infectious Agents is preserved 1. Vascular Reaction
Eosinophilic due to denaturation of proteins AND i. Vasoconstriction Occurs first and lasts only for v. Suppurative / Mainly due to bacterial infection or
enzymes seconds Purulent secondary condition
Morphological Alterations in Cell Injury Occurs on affected tissue when vessel is
obstructed, except brain ii. Vasodilation Increased diameter of blood Collection of large amount of Pus
Generalized swelling of cell and organelles - first manifestation vessels  Composed of :
Blebbing of plasma membrane Infarct - localized area of coagulative necrosis Increased blood flow to area  Neutrophil
Detachment of ribosome from ER Erythema (heat and redness on Necrotic cells
2. Liquefactive Tissue becomes liquid viscous mass due to
Clumping of nuclear chromatin site of infection) Edema fluid
digestion of dead cells
Occurs during microbial infection iii. Endothelial Increased vascular permeability 
Creamy yellow because of pus Activation Edema (extravasation of liquid Abscess = collection of pus
CELL DEATH
Affects CNS portion of blood)
- Occurs after irreversible injury 3. According to Location
2. Cellular Response
3. Gangrenous Due to ischemia and superimposed bacterial
infection i. Neutrophil WBCs enter site of injury i. Localized One site; not widespread
Difference between the Two Principal Pathways of Cell Death Activation  Kill organism, mop debris
Combination of coagulation and liquefaction
Apoptosis Necrosis necrosis  Release chemokines ii. Generalized / Whole organ; area of tissue; region
(substances that attract other Systemic of tissue
Cell Size Reduced Enlarged i. Dry Sterile necrosis
immune substances to site of
Nucleus Fragmentation Pyknosis (clumping)  Gangrene Arterial occlusion, sharp demarcation line, less foul
inflammation)
into Karyorrhexis odor
nucleosome-size (fragmentation)  ii. Wet Nonsterile necrosis (due to bacterial infection)
fragments Karyolysis (dissolution) Gangrene Vein occlusion, no sharp demarcation line, foul
Plasma Intact Disrupted odor
Sequential Steps of a Typical Inflammatory Reaction
Membrane 4. Caseous Means ‘cheese-like’ 1. The offending agent is recognized by host cells and
Cellular Intact Enzymatic digestion; may Friable white appearance of necrotic area molecules.
contents leak out of cell Seen in tuberculosis, granuloma
2. WBCs and plasma proteins are recruited from the
Adjacent No Frequent (due to leakage of
5. Fat Fat destruction due to pancreatic lipase circulation (Intravascular), and go towards the area where
Inflammation (because cellular contents)
phagocytes  Fatty Acids + Calcium = Chalky-white areas the offending agent is located.
rapidly devour the (fat saponification) 3. WBCs and proteins are activated and work together to
cells) Seen in acute pancreatitis destroy and eliminate the offending substance.
Physiologic Physiologic Pathologic 6. Fibrinoid Seen in immune reactions when antigen- antibody 4. Reaction is controlled and terminated.
or complexes are deposited in walls of arteries 5. Damaged tissue is repaired
 Immune complex + fibrin = fibrinoid (bright
Pathologic? Death by destiny Death by disease pink and amorphous appearance in H&E Note: If inflammatory reaction is not controlled, adverse tissue effects
staining)
may occur, such as abscess formation and chronic inflammation.

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ABNORMALITIES IN CELL GROWTH Classification of Tumor SOMATIC DEATH

Benign Malignant  Refers to the complete cessation of metabolic and functional
I. Retrogressive Changes - organs are smaller than the normal abilities of an organism 4. Post-mortem Clotting
Death Usually does not Usually causes death
A. Developmental Defects  Occurs immediately after death; apparent only in autopsy
cause death except
1. Aplasia - incomplete development of the organ Primary Changes of Death (CRC) Post-Mortem Clot Ante-Mortem Clot
for infants and brain
2. Hypoplasia - failure of an organ to develop fully tumors  Occurs 4-6 minutes, then death follows Upper layer is clear Has tangled,
3. Agenesia - complete non-appearance of an organ 1. Circulatory failure – start of death when cardiac function (resembles chicken irregular fibrin
Differentiation Well-differentiated Some lack of
ceases; flat electrocardiogram (ECG), and/or absence of fat); RBC settles at
4. Atresia - failure of an organ to form an opening differentiation
heartbeat is indicative Appearance the lowest part of
Rate of Growth Usually progressive Erratic; may be slow
the blood vessel
B. Atrophy - acquired decrease of the size of a normally and slow; may come to rapid 2. Respiratory failure – decrease O2 and increase CO2; loss of all (resembles currant
developed organ to a standstill or processes necessary for life; absence of respiratory sounds and jelly)
regress movements is indicative
Assumes blood Seldom assumes
Metastasis Absent Frequent Shape
II. Progressive Changes - organs become larger than normal 3. CNS failure – loss of coordination and reflexes; absence of vessel shape blood vessel shape
(spread of tumor
A. Hypertrophy - increase of cell size brain stem reflex, and/or electroencephalogram (EEG) activity is Consistency Rubbery Non-rubbery
to other sites)
1. True - due to increase work load or endocrine indicative
stimulation General Tumor Nomenclature
Secondary Changes of Death (ARLPDPA)  The next 3 stages of death occurs simultaneously and leads to
a. Compensatory - true for paired organs, where Origin Benign Malignant 1. Algor Mortis the total digestion of cells
one is excised and the other incresases in size Epithelial Tissue -carcinoma
 Cooling of the body; decrease in temperature
to “take responsibility’ for its pair Connective/ -sarcoma 5. Dessication
-oma  Equalizing of the body temperature to the external temp
2. False - due to ECF buildup and CT proliferation Mesenchyme  General drying and wrinkling of fluid-filled organs;
 Normal rate of cooling: 7OF/hr
Tissue
B. Hyperplasia - increase in cell population  Sped up by: cold environment, malnutrition/dehydration,  most evident in the cornea, and anterior chamber of eye
1. Physiologic, as in pregnancy severe hemorrhage
Example: Benign Malignant
2. Pathologic, as in typhoid fever affecting lymphoid  Slowed by: fever, extreme physical activity before death 6. Putrefaction
Epithelial lining of Adenoma Adenocarcinoma  Decomposition of body carried out by microbial action
follicles gland
2. Rigor Mortis (normal flora from gut migrates to blood vessels and
Lymph vessels Lymphangioma Lymphangiosarcoma spreads all over the body)
III. Degenerative Changes – changes in the adult form of cell  Stiffening of muscles due to lack of ATP (ATP is
responsible for driving calcium ions back to sarcoplasmic  Principal agent: Clostridium welchii (gram-positive,
A. Metaplasia - replacement of one cell type of cells to anaerobic, rod-shaped)
Grading of Tumors reticulum of muscles)
another type in the same site (reversible)  First appears in the involuntary muscles of heart  First external sign: Greenish discoloration of skin over the
B. Dysplasia – means “disordered growth”; development of - Attempts to establish an estimate of the level of  Observed in eyelids, followed by neck, then lower right iliac fossa due to bacterial hydrogen sulfide reacting
abnormal cell types within a tissue (reversible) malignancy of a tumor extremities with hemoglobin, thus forming sulphahemoglobin which
C. Anaplasia – lack of differentiation of cells (irreversible) - Based on cytologic differentiation of tumor cells and  Starts 2-3 hrs post-mortem, completes 6-12 hrs post- stains the area green
D. Neoplasia – means “new growth”; uncontrolled number of mitoses mortem; persists for 3-4 days  Eventual production of foul-smelling gas due to invasion of
 After 3 to 4 days, relaxation occurs due to breakdown of saprophytes, which leads to distension of abdomen,
proliferation of cells with no purpose; due to carcinogens, - Different from Staging, as staging accounts for size, extent
contracted muscles swelling of face and genitalia, and liquefaction of internal
or DNA alteration (irreversible) of spread to lymph nodes, and presence of metastasis organs
- Well-differentiated: tumor cells resemble normal cells  Factors: muscle activity by the time of death;
 Most resistant to putrefaction: prostate gland
 Tumor/Neoplasms – mass of neoplastic cells - Undifferentiated: tumor cells do not resemble normal cells  Sped up by: warm environment; infancy; thin-layered
muscles
7. Autolysis
Broder’s Grading of Tumors  Slowed by: cold environment; obese people
General Characteristics of Tumors  “Self-destruction”; the self-digestion of the cells by their
Grade Differentiated Undifferentiated Cells
1. May resemble and function like a normal cell own enzymes;
Cells 3. Livor Mortis/Sugillation
2. Autonomous; non-responsive to normal growth  First external sign is the whitish appearance of cornea
I 100% - 75% 0% - 25%  Purplish discoloration of skin due to blood stasis
factors
II 75% - 50% 25% - 50%  Lividity of the dependent portions of the body due to
3. Parasitic nature; competes with cells for metabolic
III 50% - 25% 50% - 75% settling of blood to the lowest parts of the body at the time
needs
IV 25% - 0% 75% - 100% of death
Parts of Tumor  Blood vessels dilate due to loss of muscle tone
1. Parenchyma – actively dividing cells  Difference of Livor Mortis from Ecchymosis References:
Limitation of Grading: Livor Mortis Ecchymosis
2. Stroma –connective tissue framework and lymphatic 1. Kumar, V., Abbas, A. K., & Aster, J. C. (2015). Robbins
and vascular channels 1. It is subjective Post-mortem stasis Trauma and Cotran pathologic basis of disease (Tenth edition).
Cause
of blood Philadelphia, PA: Elsevier/Saunder
2. Higher grades of tumor have more tendency to
After application of Discoloration No disappearance 2. Shedge, R., Krishan. K., Warrier, V., & Kanchan, T.
metastasize
pressure (Blanching disappears (2019). Postmortem Changes. In StatPearls [Internet].
3. Most sarcomas cannot be graded test) StatPearls Publishing
After incision Has oozing No oozing

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QUALITY IN HISTPATHOLOGY LABORATORY STORAGE, RETENTION, AND DISPOSAL

DAVAO DOCTORS COLLEGE
Quality Assurance Surgical Specimen
MEDICAL LABORATORY SCIENCE DEPARTMENT - ensuring that everything is right (test, time, specimen, - Storage
STUDENT NOTES: GPHCT -
patient, diagnosis & price)
Includes availability of reagents, supplies, preventive
o After grossing and cutting sections for fixation, specimen
maintenance & monitoring of equipment & evaluation of the must be returned to their original (leak-proof and airtight)
quality of services. containers, a safety measure.
HISTOPATHOLOGY Sample Workflow in the Histopathology Laboratory Quality Management Systems ■ Prevents fume formation
■ Ensures fixative is at right level
- The study of tissues affected by disease — set of coordinated activities to regulate a lab in order to
o Group specimens according to date for ease of finding
- Useful in making a diagnosis and in determining the continually improve its performance
— skilled personnel o Storage area must be free from human and animal
severity and progress of a condition
— considers pre-analytic, analytic and post-analytic phase interference.
HISTOPATHOLOGIC TECHNIQUES — proper specimen collection & processing of results & - Disposal
documentation o Placed in biohazard bags for incineration or burial
- Includes all activities done in the laboratory in order to
— highly quality of reagents & equipment
produce a suitable specimen slide for viewing by the - Returning of Specimen to Patient
— continuous professional education of staff
pathologist o Guidelines must be according to the hospital
Documents ■ Documentation of:
THE HISTOPATHOLOGY LABORATORY • Release to patient or authorized representative
- numerically, alphabetically, or chronologically arranged
• Details of specimen
• Proper info of hazards of the tissue and fixative
A. Request form
• Proper disposal
1. Name, Age, Sex, Date of Birth
o Limbs and fetuses are buried for religious purposes
2. Hospital or Lab Accession #
o Bullets, breast implants, and foreign bodies may be
3. Specimen Type/Source; Clinical Impressions
evidences for crime; released to appropriate police
4. Pertinent History, Operative Findings
authorities and with properly documented with Chain of
5. Test Requested, Procedure performed
6. Date &Time of Request, Collection and Transport
Custody (COC) form
7. Requesting Physician o Gallstones, foreign bodies, orthopedic hardware and teeth
o Respect patient confidentiality
B. Patient Report: Request Form info + Diagnosis and Gross/
Microscopic findings + Name of pathologist Paraffin Blocks and Slides
- Storage
Types of Results o Must be in cool, dry place (otherwise, paraffin may melt or
combust)
1. Surgical Pathology
2. Cytopathology
o Free from vermin and insects(may be eaten to access
HR Management tissue)
1. Pathologist 3. Autopsy Report
o Paraffin blocks may form amorphous masses that may
- Head of laboratory obscure ID
Turnaround Time (TAT) of Results o Arranged according to year and surgical pathology
2. Associate Pathologist accession number for easy retrieval
• Surgical Pathology and Cytology = 2 days
- Supports the pathologist - Request for Blocks and Slides
• Frozen Sections = 5-15 minutes
o Borrowing slides/blocks to be viewed by another
3. Histotechnologisy or Histotechnician • Autopsy Report = 7 days
institution)
- Provides slides that are properly labeled, processed, o Logbook must be available (Patient data, accession
number, purpose of borrowing, recipient, quantity, date of
stained, mounted and sequenced release and return, proper signatories, pathologist who
- Ensures that formalin and other agents are fresh and C. Telephone Report - preliminary diagnosis screened the slides)
in good working quality D. Prelim Report - status of results 48-74 hours from receiving o Slides are transported with damage and breakage
- Maintains equipment in high quality condition E. Final Report - conveys results after test is completed prevention; having insulation and shock-proof systems
- Performs preventive maintenance, as well as F. Incident Report – documents occurrence of problems
o Deposit is a customary practice. Charging for new slides
troubleshooting procedures and cuts is also practiced.
Laboratory Services o Preserve the original state of the material
1. Tissue Processing
- Disposal
2. Cytology
3. Frozen Biopsy o Regular trash (do not post health hazards)
4. Special Staining o Patient confidentiality must still be observed
5. Immunohistochemistry o Slides may be used by resident pathologists and med
students; may be used for further researches
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Retention Periods INSTRUMENTATION b) Cryostat – consists of a microtome (usually rotary), kept in a single chamber, and then fluids are pumped in and
- As suggested by College of American Pathologists (CAP), 2010 inside a cold chamber maintained at -5°C to -30°C out as required
Before tissues are mounted on slides, they should undergo different (average: -20°C); capable of freezing fresh tissues, thus
Records Storage processes. Each step in the process necessitates the use of various used for STAT diagnosis IX. Gross Table
specific components. c) Sliding – knife is moving - for gross examination and dissection of submitted
Requests, Accession logs, 2 years
I. Microscope – for tissue and cell visualization d) Freezing specimens. It should have:
Maintenance and QC logs
e) Rocking a) Sink
BB Quality Control 5 years • Bright Field Microscope – simplest and most popular f) Ultrathin b) Table Top
BB employee signatures, patient 10 years • Dark Field Microscope c) Water Supply
(Further discussed in “Trimming and Microtomy” topic) d) Irrigation System
records, donor and recipient records - For unstained and transparent samples
Only oblique rays hit the object e) Fume Ventilation
Records of indefinitely/permanently Indefinitely - III. Microwave Oven
deferred donors; forensic accession - Samples are made brightly lit against dark background f) Waste Disposal Unit
- best for antigen preservation
logs • Phase Contrast Microscope - used in epitope retrieval for Immunohistochemistry
(Further discussed in “Gross Examination” topic)
- Phase shifts in light passing through transparent and - used in speeding up procedures
colorless specimen are converted into brightness - agitation and heating will increases fixation rate X. Others
Specimens Storage (contrast) changes in the specimen, making them 1) Wares: Coplin jars, staining racks, staining dishes (all
visible Others: Incubator Oven three are for staining) and other glass and plastic materials
Urine 24 hours - does not require staining - in situ hybridization and enzyme reactions (for storage, and preparation of solutions)
- Thermal Requirements: 37°C 2) pH meters - measurement of solution and buffers
Serum and other body fluids 48 hours • Polarizing Microscope
3) Grossing tools – for gross examination
- Designed to examine birefringent properties of IV. Automated Stainers
Microbiology and blood 7 days 4) Freezer (Thermal Requirement: -20°C)
anisotropic specimens - Eliminate the need for the laborious manual staining
smears (routine) 5) Refrigerators (Thermal Requirement: 4°C)
- Birefringence: splitting of one ray of light into two - May operate through:
BB donor/recipient (blood) 7 days post-transfusion - Anisotropism: substances that exhibit different o Dipping the slide into the stain; and/or
properties when measured in different directions o Applying the stain to the slides
Surgical Tissues 2 weeks after final report - NOTE: Amyloid in Congo Red has Apple Green
Cytogenetic slides 3 years birefringence V. Slide Dryers
- removing water collected during sectioning (water from
Cytology slides (e.g. pap) 5 years • Fluorescence Microscope flotation bath)
- Uses ability of substance to exhibit fluorescence - Thermal requirement: 5-10°C HIGHER than the melting
Tissue, BM, FNA slides 10 years
- Fluorescence: emission of low frequency light of point of paraffin
Paraffin blocks 10 years substances when they are illuminated with high - NOTE: Overheating causes uneven staining, artifacts
energy light formation and tissue destruction
Forensic: Blocks, Slides Indefinitely - Only allows observation of the specific structures
which have been labeled for fluorescence VI. Flotation Bath
- NOTE: Acridine Orange stains DNA (nucleus) - - fishing out of tissue section; keeps sections from wrinkling
yellow, and RNA (cytoplasm) – orange - has “black” interior, for easy visualization of sections
Reports (results) Storage - Thermal requirement: 5-10°C LOWER than the melting
• Electron Microscope point of paraffin
Clinical pathology (e.g. CC, Hema) 2 years
- Uses a beam of accelerated electrons as source of
Anatomical/Surgical pathology 10 years illumination VII. Embedding Centers
- Has higher resolving power than light microscopes - System designed for paraffin embedding
Cytogenetics (final reports and 20 years and can reveal the structure of smaller objects - Thermal requirement: 2-4°C higher than melting point of
photographs)
II. Microtome – for producing tissue ribbons or sections paraffin
Forensic autopsy Indefinitely Main Parts: Components:
a) Block Holder a) Paraffin dispenser and reservoir
b) Knife Holder b) Orientation stage
c) Pawl and Feedwheel Mechanism c) Chilling plate
d) Warm storage (for embedding mold)
Kinds of Microtome
[Things to know: Thickness of the tissue produced, Inventor VIII. Automated Tissue Processor
Microscope to be used, Embedding Medium] - may be stationary or moving
- equipped with alarm and warn technicians if high temp
a) Rotary – most commonly used; for routine and serial - others use vacuum in heat to speed up procedures
(continuous) sections; knife is stationary
Thickness: 3 to 5 micrometers Main Types
Inventor: Minot a. Tissue-Transfer Machine (“dip and dunk”): specimens are
Microscope: Light Microscope transferred from container to container
Embedding material: Paraffin b. Fluid-Transfer Machine (“enclosed”): specimens are held
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EXAMINATION OF TISSUES
Tissues for examination are usually obtained through biopsy, or autopsy. They range from whole organs or very large specimens to tiny fragments of strands are teased apart using an application stick FIXED TISSUE EXAMINATION
tissue. • ADV: maintains intercellular relationships
• For fresh sputum, bronchial aspirates, and thick mucoid - The end goal is to produce a tissue section of good quality that
Specimens Received secretions allows for adequate interpretation of microscopic cellular
changes (for diagnosis)
iii. Pull-apart
- Accomplished by fixing the tissues and carefully processing
• A drop of the material is placed into a clean slide, and
Biopsy Autopsy them to preserve their structures, then impregnating them with
covered with another clean slide. Material is allowed to
hardening substance to permit making thin slices suitable for
disperse evenly
staining and microscopic evaluation
Methods of Examination Dissection Techniques • Slides are then pulled apart with a single, uninterrupted
movement in opposite directions
Tissue Processing
Fresh Fixed Virchow Rokitansky Ghon Letulle iv. Touch Prep - various steps required to take the tissue from fixation to the
• AKA Impression Smear state where it is completely infiltrated with a suitable
Frozen • Surface of a freshly cut tissue is pressed to a clean slide histological wax and can be embedded ready for section
Teasing Crushing Smear Prep • For Phase-Contrast Microscopy cutting on the microtome
Sections
• ADV: maintains intercellular relationships 1. Fixation
4. Frozen Sections • Preservation of tissue constituents in a life-like manner as
Streaking Spreading Pull-apart Touch Prep possible
- For rapid diagnosis of tissue
- Requested during intra-operative procedures to help 2. Decalcification (optional)
BIOPSY FRESH TISSUE EXAMINATION surgeon in choosing his next plan of action • Removal of calcium or lime salts from bones following
- Recommended for demonstration of lipids and nervous fixation
- Ante-mortem examination of tissues - Allows examination of cells in their living state • For ease of cutting
tissue
(ante=before; mortem=death) - ADV: View protoplasmic activities (motion, mitosis, - Fresh tissues are frozen using a cryostat or freezing 3. Dehydration
- Examination of tissue sample from the living phagocytosis, pinocytosis) microtome • Removal of water
- DADV: Subject to ischemia, therefore not permanent, and liable - ADV: rapid processing time with less equipment 4. Clearing/Dealcoholization
Types of Biopsy (but not limited to the following) to changes requirement, and less need for ventilation • Removal of the dehydrating agent with the use of a
Fine needle Simplest, least invasive - DADV: relatively poor quality of the final slide; expensive clearing agent (a solvent miscible with both the dehydrant
Methods
aspiration (FNA) Uses very thin needle attached to syringe to take (Further discussed in “Rapid Processing Techniques” topic) and impregnating material)
out small amount of fluid and tissue from area 1. Teasing
- AKA Dissociation 5. Infiltration/Impregnation
- Selected tissue is immersed in petri dish/watch glass • Replacement of clearing material with impregnating
Core needle Uses slightly larger needle material
Remove small column of tissue (1/16 inch in containing isotonic solution, and then carefully dissected
• Gives firm consistency to tissue for ease of handling and
diameter, ½ inch long and separated using needle or applicator stick
sectioning
- Tissue is then transferred to slide and examined under the
Incisional Surgical; Small part of a large lesion or tumor is
microscope (phase contrast or bright field) 6. Embedding
taken • Placing the infiltrated tissue into a mold filled with molten
- May be stained using supravital dyes
Excisional Surgical; Entire affected area is taken wax to form a solid tissue block
2. Squash Preparation
Punch For skin; Uses circular blade to obtain deeper skin - AKA Crushing 7. Trimming
sample that removes a short cylindrical core of - Small pieces of tissue with diameter less than 1mm are  Removal of excess wax from tissue block, in preparation
tissue (“apple core”) for microtomy
compressed between two slides
- May be stained using supravital dye 8. Section-Cutting/Microtomy
Shave For skin; small fragments of outer layers of skin
• Cutting of tissues into fine tissue sections with the use of a
are “shaved” or scraped 3. Smear microtome
- Method depends on nature of material to be examined
Currettage Tissues are removed from body cavity (or canals) 9. Staining
- Useful for cytological examinations
using a curette (instrument with a tip shaped like a
- Cellular materials are spread over a slide using wire loop,  Application of dyes to sections for visualization
small scoop or hook)
applicator stick or slide
10. Mounting
- Vital stains may also be applied  Use of media to mount a coverslip for ease of handling,
Methods of Examination of Biopsy Specimens - May be made permanent by fixing them storage and protection of sectioned tissue
Fresh i. Streaking 11. Labelling
• Rapid, but gentle zigzag application of the material  Indicating of accession number and year for proper
Fixed (Preserved)
throughout slide identification
• Must have a relatively uniform distribution
ii. Spreading
• Material is gently spread onto the slide, and the mucous
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DAVAO DOCTORS COLLEGE

AUTOPSY Proper Handling of Autopsy Specimens
MEDICAL LABORATORY SCIENCE DEPARTMENT
-
-
AKA Necropsy; Thanatopsy
Post-mortem examination of tissues
1. Organ block removed from the body cavity should be
thoroughly washed of blood using cool or cold water to STUDENT NOTES: GPHCT
minimize the blood staining of organs. Never use hot water.
Purposes 2. Organ blocks are placed in a large enameled pot containing
fixative, filled to about one third capacity. PRE-ANALYTICAL FACTORS
- Determine cause of death and extent of injury Specimen Accessioning
3. Tissues should not be pressed against each other or the
- Uncovering existence of an undetected disease Ischemia Time - 1st and most important step in HP outside the tissue processing
bottom or walls of the container.
4. Lesions that are encountered during dissection should be - Time interval between surgical intervention and proper fixation of procedures
Types of Autopsy according to: the removed specimen - Specimens are given a unique identification number (may be a
obtained early and placed in fixative before organ is fully
Medical/Hospital – performed on a patient - If prolonged, ischemia will allow activation of tissue enzymes, barcode, for some laboratories) that will identify each specimen
 incised.
who dies in a hospital autolysis, and degradation of proteins and nucleic acids, which for each patient
during course of affects visualization of tissues - Also useful for ease of retrieval of specimens, slides, and blocks
treatment - Divided into: - Indicating codes may be used for the following
Purpose  Medico-legal – generates evidentiary o Surgical
document that forms a basis a) Warm Ischemia Time o Autopsy
for opinions rendered in a References: - Occurs during operation when blood supply of tissue is cut o Cytology
criminal trial, civil suit and the 1. Bruce-Gregorios, J. H. (2016). Histopathologic techniques. off - Sample Format of Accession Number:
like (2nd Revised Edition). Makati, PH: Katha Publishing - During this period, the tissue is alive and active, but will Indicating Code – Year – ID Number of Specimen
(611.0182/B83) undergo progressive metabolic stress due to hypoxia E.g. #S20-12345
Completeness  Partial 2. Lo, R., Orillaza, M., Madrid, M., Santiago, F., & Aguilar, P. - Affected by the whole surgical procedure (complexity of
 Complete
(2015). Basic histopathologic techniques. Metro Manila, procedure, ability of surgeon, modality of intervention) - Avoid serial accessioning of similar specimen types to reduce
 Y-shaped PH: C & E Publishing - Beyond the control of the histopathology lab mix-up of specimens, and cross-contamination.
Manner of Incision  Straight Cut (I-shaped) 3. Waters, B. L. (Ed.). (2010). Handbook of autopsy practice. E.g. gastric biopsies are interspersed with other tissue types
Springer Science & Business Media b) Cold Ischemia Time
- Interval between tissue removal from the patient and arrival
in the pathology laboratory for grossing
Dissection/Evisceration Techniques - If prolonged, temperature of specimen will gradually reach
Virchow - One by one removal of organs the external temperature, and autolysis and drying of the
- most widely used
surface may occur
Rokitansky - "in situ" (in place) dissection, followed by en bloc - Extensions may contribute to poor fixation
removal
Pre-Analytic Fixation
Ghon - "en bloc" removal
- All parts to be examined must be initially fixed
- Organs of same group/cavity/region are
- Earlier fixation  better preservation of tissue morphology
removed at the same time
- Improper fixation may impede processing (poor fixation has no
Letulle - "en masse" removal of organs remedy
- All organs are removed at the same time, then - Proper ratio with tissue must be observed
dissected by blocks o 3-5mm thick tissues may be fixed for 6-48hrs
o 5mm thick tissues and large tissues (such as limbs) must be
sectioned prior to fixation, or else, fixation will not be complete
Prerequisites to Performing Autopsy and may occur only at the periphery of the tissue
 Written or informed consent from the legal next-of-kin
Order of priority: spouse, adult child, either parent, adult Specimen Reception
sibling, grandparent, guardian - Submitted specimens must be put in a container labeled with
 Medical abstract or clinical data patient’s name and specimen source/site, and must be
 Autopsy Request (suspicious evidence of foul play) accompanied with a duly accomplished pathology requisition
form.
Personnel
 Coroner – a public official who is empowered to order an - Criteria for Rejection of Specimens:
a) Discrepancies between requisition form and specimen
inquest into the manner or cause of death
labels
 Prosector – pathologist who performs the dissection b) Unlabeled, mislabeled, and inappropriately identified
 Diener – comes from German word “leichendiener” meaning specimens (last resort: DNA identification)
“servant of the dead”; assists during autopsy, and assumes c) Leaking specimen containers
many and varied responsibilities in the autopsy laboratory d) Absent clinical data or history, and other necessary info

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GROSS EXAMINATION characteristics. Examples are hyperplastic tissues.) Hollow Structures

 Dimensions (length, width, depth) are rounded to nearest 1.0 cm.
- Consists of describing the specimen and placing all or parts of it For multiple pieces, indicate size of the largest piece. - Must be cut open longitudinally and fixed with cottons inside
Specimens for Gross Description Only
into a plastic cassette, in preparation for tissue processing (because disease is not histologic level)
- One of the basis of pathologists' diagnosis Sectioning Lymph Nodes
- Involves selection of elements that appear to be of clinical  Accessory digits  Prosthetic breast implant - Taking a representative sample of the tissue. - most important component of tumor resections because they are
significance for histologic examination  Bunions (aka hallux valgus) &  Prosthetic heart valves essential for prognosis and planning therapeutic options
hammer toes without attached tissue Small Cut serially about 2 mm thick to look for small - Should be received fresh and not immersed in formalin
Cutting Tools: cleaned before and after use (to avoid carryover)  Extraocular muscle from  Tonsils and adenoids from specimens lesions. Lesions are then sampled for - Node is bivalved, and entirely submitted
o Scissors corrective surgery children histologic exam. Filter paper may be used in - Sentinel lymph nodes: usually first lymph node to be involved
o Forceps  Inguinal hernia sacs in adult  Umbilical hernia sacs in wrapping small samples during metastasis. Entirely submitted. However, large specimens
o Blade holders  Nasal bone & cartilage from children Large Cut at an interval of 1 cm thickness (termed may be bisected, and submitted in one or two cassettes
o Blades - disposed in sharps container rhinoplasty  Varicose veins specimens as breadloafing) to ensure that pathologic
Mastectomy
areas or tumoral areas are identified
Gross Table or Gross Workstations - Note for weight, size of breast and axillary dissection, skin ellipse,
 Sink nipple scar, basal margins
 Tabletop Specimens that may be excluded from mandatory
submission to Histopathology laboratory  Indicate number of sections and blocks on the gross description Pediatric SPX
 Water supply  Specimen must fit easily into the standard cassette, which
 Irrigation system • Bone donated to the bone bank
measures 3 x 2.5 x 0.4 cm - Additional processes such as IHC, flow cytometry, cytogenetics
 Fume extraction/ventilation system • Bone segments removed as part of reconstructive orthopedic
 Thickness: not more than 0.3 cm to allow for closing of cassette and molecular genetics is often done. These may require fresh,
 Water disposal unit procedures frozen, or specially processed tissues
• Cataracts removed by pharcoemulsification
and fixative penetration
• Dental appliances and teeth with no attached soft tissue
 When possible, edges of tissue should be squared
Specimen Categories  Paper tags are embedded in the cassette. They should labelled Specimen with Tumor
• Fat removed by liposuction - Identify:
A  Specimens only requiring transfer from container to with accession number using pencil. Markers and pens will
• Foreign bodies such as bullets or other medico-legal evidence ► Site & size of tumor
tissue cassette. No dissection required given directly to law enforcement personnel dissolve upon processing.
► Location & structure invaded by tumor
 May need to be placed in filter paper first before • Foreskin from circumcision
 If printed, dot matrix must be used.
► Vascular invasion
placing in cassette because of their small size • IUDs without attached soft tissue
 Original containers with specimen are saved until case is signed
► Presence of lymph node
• Medical devices (catheters, gastrostomy tubes, stents, and
out (backup evidence in cases of discrepancies) ► Distance from resection margin
e.g. endometrium, colonic series, breast core biopsies sutures) that have not contributed to the patient’s illness, injury,
Other Specimen Considerations
or death
B  Specimens requiring transfer and routine sample • Middle ear ossicles Brain Specimen Worksheet
dissection: sampling, counting, weighing, or slicing • Orthopedic hardware and other radiopaque medical devices, - Aka “gross worksheet”
- Brain is fixed first before grossed
provided that there is an alternative policy for documentation of - Guides histotechnician in assuring that all blocks are processed
 Tied at the Circle of Willis and suspended
e.g. small lipoma, small skin biopsy, cervical LLETZ their surgical removal  Must not touch side of container to avoid deformity - Must be properly filled up; filled for future reference
• Placentas that do not meet institutionally specified criteria for - Contains the following:
(Large Loop Excision or Transformation Zone)  In 10% NBF for 2-3 weeks
examination • Accession number
C  Simple dissection required with sampling needing • Rib segments or other tissues removed only for purposes of Colon Cancers • Number of sections and blocks
a low level of diagnostic assessment and/or gaining surgical access, provided that the patient does not have - Polyps: Base (the area where cautery arteries are located) is • “Comments” column (for special requests & etc.)
a malignancy always inked. • Gross description
preparation
• Saphenous vein segments harvested from coronary artery o Small polyps: Bisected and placed in one cassette
bypass
e.g. Prepuce (fore skin in male) / Folds in clitoris o Large polyps: sides are trimmed away from the stalk,
• Skin or other normal tissue removed during cosmetic or and stalk is placed in a separate cassette
(female) Gall bladder, hemorrhoids, appendix reconstructive procedure (not a lesion or the patient does not
D  Dissection and sampling required needing a have a history or malignancy) Dermatologic SPX References:
moderate level of assessment • Therapeutic radioactive sources 1. Bruce-Gregorios, J. H. (2016). Histopathologic techniques.
- Vertical orientation is always maintained (using markers)
• Normal toenails and fingernails that are incidentally removed (2nd Revised Edition). Makati, PH: Katha Publishing
- Punch biopsies are submitted whole
e.g. Pigmented skin lesions, skin w/ markers, large (611.0182/B83)
Describing Specimens and Gross Description - Tissues greater than 4mm are dissected
2. Lo, R., Orillaza, M., Madrid, M., Santiago, F., & Aguilar, P.
intestine (Crohn's), large glands tumors - Identify the specimen. Note and verify all anatomical structures. - Skin ellipses: serially cut along the short axis at 2 to 3 mm interval.
(2015). Basic histopathologic techniques. Metro Manila, PH:
E  Specimens requiring complex dissection and - Identify orientation markers used by surgeons, if available The two most distal sections or tips are submitted in two separate C & E Publishing
sampling methods a. Inks – used to identify and orient the specimen’s cassettes. Remainder is submitted in one or more cassettes
components, distinguish samples, for embedding
instructions Eyes
e.g. thyroid, breast cancer, testis (seminoma), uteri b. Nicks – indicates laterality - Inject fixative first then gross.
c. Sutures – represented by LL: long lateral; or SS – short
superior Hard Tissues
- Describe all notable characteristics: type of specimen, shape, - Wash in running water then immerse in tissue softeners
color, texture, consistency, dimensions, weight
 Weight of intact organs are rounded to the nearest 0.1g. (In
some cases, weight is more important than histopathologic
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DAVAO DOCTORS COLLEGE

TYPES OF FIXATIVES I. ALDEHYDES
MEDICAL LABORATORY SCIENCE DEPARTMENT According to Composition 1. Formaldehyde AKA Formalin
STUDENT NOTES: GPHCT A. Simple made of one component  For routine HP techniques
 Produced from oxidation of methanol
i. Aldehyde  Usually buffered to pH 7 with phosphate buffer
a. Formaldehyde
FIXATION Factors Affecting Fixation b. Glutaraldehyde Concentrations:
- First and most critical step in tissue processing because if fixation 1. Fixative of  10% Neutral Buffered Formalin (NBF) ii. Metallic Fixatives
Choice o 100% - gas form
is inadequate, the succeeding tissue processing steps will also  Morphologic criteria for diagnosis have a. Mercuric chloride
be inadequate o 37-40% - stock concentration (causes overhardening
been established based on Formalin- b. Chromate
- Primary purpose: of the external surfaces of tissues)
Fixed Paraffin Embedded Specimen c. Lead
 Preserve morphological & chemical integrity of cell in a life- o 10% - working solution; most commonly used
(FFPES) iii. Picric acid
like manner as possible by stopping all cellular activities iv. Glacial acetic acid
2. Time  Fixation must be done 20-30mins after  ADV: cheap, readily available, easy to prepare, stable,
- Performed as soon as tissue is removed from the body v. Alcohol
cutting off blood supply (to shorten compatible with most stains
- If tissues/cells are exposed to: warm ischemia time) vi. Osmium Tetroxide
a. Air  drying of tissue  DADV: nose and eye-irritant, may cause allergic dermatitis
 Prolonged fixation shrinkage vii. Trichloroacetic acid
b. Water  swelling of cells viii. Acetone
c. Saline  shrinkage of cells ix. Heat Effect Cause Remedy
3. Tissue-to-  1:10 or 1:20 (tissue to fixative ratio) White Prolonged  Filter o
Fixative Ratio  1:20 is more common paraformaldehyde storage  Add 10% methanol
 Osmic acid fixatives: 1:5
B. Compound 2 or more components or fixatives
Effects of Fixatives precipitatesturbidity (but denatures
■ Hardens soft tissues in preparation for further tissue processing proteins thus
■ Render cells resistant to damage caused by chemicals used in 4. Penetration Rate  Formalin: 1 mm/hr (but slows down as unsuitable for EM)
it goes deeper into the tissue) According to Action
further processing A. Microanatomical (10,10,HFZZBB)
■ Inhibit decomposition caused by bacteria and fungi 5. Thickness of  Larger  Longer fixation time, more Formation of formic Unbuffered  Buffer
Section permits general study of tissues without altering the
■ Minimize the risk of occupational infection fixative acid  reduced  Methanol
structure of the subjects of interest
■ Act as mordant for certain stains, thus promoting or hastening  Light Microscopy: 2cm2 x 0.4cm staining quality, and
1. 10% NBF
staining, or inhibit certain dyes  Electron Microscopy: 1-2 mm2 formation of formalin  10% formol saline +
2. 10% Formol-Saline
pigment Mg++/Ca++ carbonate
3. Heidenhain’s Susa
Characteristics of Ideal Fixative 6. Tissue  Longer fixation time: in jar with marbles
4. Formol-Sublimate/Corrosive
1. Cheap Components Fibrous tissues
Presence of Mucus (wash with NSS) 5. Zenker’s
2. Stable Brown/black Action of  Saturated alcoholic
Fat (cut into thin slices fixed longer) 6. Zenker-formol (Helly’s)
3. Safe to handle precipitates formic acid picric acid
Blood (flushed out with saline) 7. Bouin’s
4. Kill cells quickly to minimize cell distortion with excess  1% KOH in 80%
8. Brasil’s
5. Inhibit bacterial decomposition and autolysis  Shorter fixation time: blood ROH
6. Permit rapid and even penetration of tissues Small or loosely textured tissues B. Cytological
7. Must harden tissues thus easier cutting of tissues  Kardasewitsch’s
preserve specific parts of the cell
8. Must make cellular components insoluble to hypotonic 7. Hydrogen Ion  Optimal pH: 6 to 8 Method
1. Nuclear Fixatives 2. Cytoplasmic Fixatives
solutions, and insensitive to subsequent processing Concentration  If outside this pH, ultrastuctural (70% ETOH & 28%
o Preserve nucleus o Other organelles aside
9. Permit application of staining procedures (pH) changes may occur ammonia H20)
from nucleus
 May require the use of buffers
o pH ≤ 4-6 o pH > 4-6
NOTE: No single fixative has all the mentioned characteristics. Each  For Electron microscopy: pH should  Lillie’s MTD
o Glacial acetic acid o HAc destroys
of them has own advantages and disadvantages. match physiologic pH (Acetone, H2O2
has affinity to mitochondria and Golgi
8. Temperature - Higher temp  faster fixation rate and
bodies 70% ETOH & 28%
Mechanism of Fixation autolysis nuclear chromatin
ammonia water)
1. Additive Fixative will forms cross-links between - Cold temp  enzyme inactivation
a. Flemming’s with a. Helly’s
Fixation soluble molecules, thus gluing them Room temp to 45OC - Optimal Temperature (routine)
glacial acetic acid b. Orth’s
together into an insoluble meshwork 40 OC - Tissue processors 2. 10% Formol-Saline
b. Carnoy’s c. Regaud’s/Moller’s
Up to 65OC - Microwave processing  Formalin diluted with 10% NaCl
2. Non-additive Fixative will not chemically bind with tissue c. Bouin’s d. Formalin with Post-chroming
0-4OC - Electron microscopy  Traditionally, the most common fixative
Fixation but removes water from tissue protein d. Newcomer’s e. Flemming’s without glacial
100OC - Tuberculosis  Recommended for CNS tissue and general post-mortem
groups thus causing denaturation of cell e. Heidenhain’s acetic acid
60 OC - Rapid biopsy tissues for histochemical examination
proteins
(FCBNH) (HORFF)  ADV: ideal for Silver impregnation staining technique
9. Osmolality  Hypertonicitycell shrinkage  DADV: tissue shrinks during alcohol dehydration [Remedy:
C. Histochemical
 Isotonicity and hypotonicity cell Secondary fixation]
Preserves chemical constituents of cells & tissues
swelling 1. 10% Formol Saline
 Thus, maintain tissues at slightly 2. Absolute ethanol 3. 10% Neutral Buffered Formalin (NBF) or Phosphate
hypertonic solution (400-450 mOsm) 3. Newcomer’s Buffered Formalin
4. Acetone  pH 7
10. Agitation,  Hastens fixation (10FANA)  Best general tissue fixative
Vacuum
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 Best for iron-containing pigments and elastic fibers which II. METALLIC FIXATIVES 2. Chromate Fixatives IV. GLACIAL ACETIC ACID
do not stain well after Susa, Zenker or Chromate fixation, Mercuric chloride Zenker  Incorporated in compound fixatives
 DADV: longer to prepare, inert to phospholipids and neutral (ZZCHBS) Zenker-Formol (Helly’s) a. Chromic acid  Solidifies at 17OCI
fats Carnoy-Lebron  Conc.: 1-2% aqueous solutions  Important for nuclear fixatives (precipitates nucleoproteins,
Heidenhain’s Susa  Precipitates all proteins, and preserves carbohydrates chromatins)
4. Formol-Sublimate/Corrosive B5  Destroys mitochondria and Golgi elements, thus not for
 Has HgCl2 Schaudinn’s b. Regaud’s/Muller’s cytoplasmic fixation
 ADV: Excellent for silver reticulum staining method, does  P: chromatin, mitochondria, mitotic figures, golgi
not need washing, fixes lipids Chromates Chromic acid bodies, RBC and colloid-containing TSEs
 DADV: forms mercuric chloride deposits (CROP) Regaud’s/Muller’s  DADV: prolonged fixation may lead to blackening of V. ALCOHOL FIXATIVES
Orth’s tissue pigment  ADV: good for glycogen
5. Gendre’s (Alcoholic Formalin) Potassium dichromate [Remedy: Wash in running tap water before  DADV: never for FATs and LIPOPROTEINS (dissolves); causes
 Has 95% ETOH, Picric acid, and GHAc dehydration] polarization of glycogen (granules will move towards the poles
 ADV: good for microincineration techniques, fixes sputum Lead or ends of the cells)
c. Orth’s  Effect: rapidly denatures and precips CHONs, preserves nuclear
6. Hollande’s  P: early degenerative processes and necrosis, stains
 For gastrointestinal (GI) tissues, prostate biopsies, and 1. Mercuric Chloride (HgCl2) demonstration of Rickettsia and other bacteria  (CEMING)
bone marrow (BM)  Most common metallic fixative  E: preserves myelin
 A: penetrates and hardens tissue rapidly 1. Carnoy’s Fixative
7. Glutaraldehyde  Routine fixative of choice for preservation of cell detail in d. 3% Potassium dichromate  Most rapid tissue fixative
 Made up of 2 formaldehyde resides linked by three carbon tissue photography  E: preserves lipids, mitochondria, at pH4.5-5.2,  Fixing brain tissues for rabies diagnosis
chains  Conc. 5-7% cytoplasm, chromatin and chromosome are fixed  E: fixes Nissl granules (Tigroid substance) and cytoplasmic
 For enzyme histochemistry and electron microscopy  Mostly incorporated in compound fixatives  Corrosive, thus avoid skin contact granules
 ADV: more pleasant and less irritating compared to  DADV: Banned worldwide d/t extreme toxicity, marked cell
formalin shrinkage [Remedy: add acid] 3. 4% Aqueous Lead 2. 70-100% Ethanol
 DADV: less stable and more expensive than formalin  May produce black granular deposits except in  P: for acid mucopolysaccharides and mucin  Enzyme studies
 Container must be refrigerated Heidenhain’s Susa  DADV: Prolonged standing  formation of insoluble lead  Does not fix but preserves glycogen
carbonate
Concentrations: Remedy: Dezenkerization [Remedy: add drops of acetic acid to dissolve residue] 3. 100% Methanol/Wood alcohol
o 0.25% - for immunoEM 0.5% Iodine + 70% ETOH  H20  5% Na thiosulfate  H20  Dry and wet smears, BM smears, bacterial smears
o 2.5% - for small TSE fragments
o 3% - most common a. Zenker’s III. PICRIC ACID
4. 95% Isopropyl Alcohol/Rubbing Alcohol
o 4% - for large TSE  HgCl2 + potassium dichromate + glacial acetic acid  Used in strong saturated aqueous solution (1%)  Touch prep smears to be Wright-stained
 Good general fixative for adequate preservation of all  For Glycogen preservation
8. Paraformaldehyde kinds of tissues  ADV: may be used as a stain as yellowing of tissue will prevent 5. Newcomer’s
 Polymer of formalin  Good for Trichrome staining small fragments from being overlooked; suitable also with  Mucopolysaccharides and nuclear CHONs
 Powder in form, used in 4% Aniline stains  Better reaction in Feulgen stain than Carnoy’s
 Plastic embedding b. Zenker-formol/Helly’s  DADV:
 For ultrathin and electron microscopy - HgCl2 + potassium dichromate + strong formalin (40%) 1. Explosive when dry 6. Gendre’s (Alcoholic Formalin)
 For piituitary gland, BM, blood-containing organs, [Remedy: add distilled H2O or 0.5-1% saturated alcohol]
9. Karnovsky’s Paraformaldehyde/Glutaraldehyde preserves cytoplasmic granules 2. Yellowing of tissues  excessive staining
 Acrolein in glutaraldehyde or formalin  Brown pigments are removed with saturated alcoholic [Remedy: immerse in Li2CO3 with 70%ROH  water  VI. OSMIUM TETROXIDE / OSMIC ACID
 P: for Electron Histochemistry and Electron picric acid or NaOH 70% ethanol  5% Na thiosulfate  water]  Pale yellow powder in water (6% in 20OC)
Immunocytochemistry 3. RBC hemolysis
 Ultrathin sections in Electron Microscopy
c. Heidenhain Susa  (PBB)
 E: Fixes and stains conjugated fats and lipids black
10. 40% Aqueous Glyoxal  Susa: Su = sublimate ; Sa = saure (acid)  DADV: very expensive, very volatile, inhibits hematoxylin
 ADV: no smudging of nuclei and distortion of staining  HgCl2 + NaCl + TCA + glacial acetic acid + formalin 1. Bouin’s
 Tissue-to-fixative ratio: 1:5
compared with formalin  Skin tumor biopsy  P: for embryo and pituitary biopsies, and tissues to be
stained with Masson’s Trichrome  (OFF)
 D: reduced staining capacity  ADV: minimum cell shrinkage and tissue hardening
[Remedy: increase staining time] due to counter-balance effect of acids and mercury:  ADV: minimum cell shrinkage and tissue hardening due to
1. Flemming’s
Acids : swelling counter-balance effect of glacial acetic acid (swelling) and
picric acid (shrinking)  Most common osmic acid fixative
Mercury: shrinkage  P: nuclear structures
 Does not produce black pigments  DADV: poorly penetrates large tissue, thus limited to small
fragments of tissues  Effect: permanently fixes fat
 DADV: Weigert’s staining of elastic fibers not possible  ADV: needs less amount of fixative
d. B5 Fixative 2. Brasil’s
 C: TCA 2. Flemming’s w/o acetic acid
 HgCl2 + Anhydrous Na acetate  Cytoplasmic structures
 BM biopsies  ADV: Better and less messy than Bouin’s

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DAVAO DOCTORS COLLEGE

VII. TRICHLOROACETIC FIXATIVES IMPROPER FIXATION
Effect Reason
MEDICAL LABORATORY SCIENCE DEPARTMENT
 Incorporated also in compound fixatives
 Marked swelling effect on tissues 1. Failure to arrest early
cellular autolysis
Failure of fixing immediately
or insufficient fixative
STUDENT NOTES: GPHCT
 Poor penetrating agent thus for small pieces of tissues or bones
 Weak decalcifying agent, thus has softening effect on dense
fibrous tissues
DECALCIFICATION ACIDS
VIII. ACETONE 2. Too brittle and too hard Prolonged fixation - Follows fixation - Most widely used, stable, easily available, relatively inexpensive
blocks or tissue - Removal of calcium or lime salts from calcified tissues
 Used at cold temp -54OC
- Inadequate decalcification  poor cutting of hard tissues an 1. Nitric acid
 For water-diffusible enzymes (Phosphatase, Lipase) 2. Hydrochloric acid
3. Shrinkage and swellings damage to the knife edge during sectioning
 For brain tissues (such as in Rabies) of cells in tissue blocks 3. Formic acid
- Hastened by heat and agitation 4. Trichloroacetic acid
 DADV: Dissolves fat, evaporates rapidly With a common 2-day duration
- 5. Sulfurous acid
4. Soft and feather-like Insufficient or incomplete 6. Chromic acid
tissues fixation - Calcifications cause a grating sensation during sectioning 7. Citric acid
IX. HEAT
5. Presence of artefact Insufficient washing of [Remedy: Remove block from the chuck, and place face down 1. Nitric acid
 Principle: Thermal coagulation of tissue proteins pigments on sections fixative on cotton or gauze with 10% HCl
 For rapid diagnosis: frozen tissue sections and Bacterial smear - Most common and fastest agents
6. Enzyme inactivation and Wrong choice of fixative - Hematoxylin-stained microcalcifications = dark purple granular - Removed by 70% ROH during dehydration
prep loss masses with light purple halos
- Imparts yellow coloration d/t nitrous acid formation
7. Removal of fixative [Remedy: add Urea or Sodium thiosulfate/sulfate]
Microwave Technique soluble substances Done on:
 PCPL: Increases movement of molecules to accelerate  Bone
fixation, staining, decalcification a. 10% Aqueous Nitric Acid
 Teeth  Urgent, needle and small biopsies
 Electron Microscopy and immunohistochemistry  Teratoma (means monster)
 ADV: Tissue is heated right through the block in a very  Calcified tissues: tuberculous organs, arteriosclerotic vessels
short time; preserves neurochemical substances b. Formol-Nitric Acid
(acetylcholine)  Has formalin (allows less destruction)
Grossing:  Urgent biopsies
 DADV: Penetrates at 10-15mm thickness; spores and References:  Done before fixation
pathogen may remain in tissues 1. Bruce-Gregorios, J. H. (2016). Histopathologic techniques.  Double gloves, eye protection glasses, face mask c. Perenyi's fluid
 Optimum Temp: 45-55OC (2nd Revised Edition). Makati, PH: Katha Publishing  Specialized table for bone processing  Has chromic acid and ROH (thus, no tissue breakup)
(611.0182/B83)  Bone specimens are grossed in fresh state  Also a tissue softener
2. Lo, R., Orillaza, M., Madrid, M., Santiago, F., & Aguilar, P.  Bone dust particles can be loaded with blood or infectious
SECONDARY FIXATION (2015). Basic histopathologic techniques. Metro Manila, PH: malts (osteomyelitis, gangrene) d. Phloroglucin Nitric acid
C & E Publishing  Fine fret-saw saw  hand razor  Most rapid decalcifying agent
 “Refixation” with another fixative
 Complete decalcification cannot be determined
o Done before dehydration or restaining of deparaffinized
Factors Affecting Decalcification through chemical means
TSEs
1. Concentration =faster, but may be more harmful to  Removed with 3 changes of 70-90% ETOH
o Improve demonstration of substance tissue
o Make special staining techniques possible (with the next 2. Tissue-to-volume optimum: 1:20
fixative as mordant) ratio 2. Hydrochloric acid
o Ensure further and complete handling 3. Temperature =faster, but may be more harmful to - Slower and causes more distortion compared to HNO3
 Post-Chromatization tissue - Provides good nuclear staining
o Use of 2.5-3% aqueous K2Cr2O7 that will act as mordant optimum: 18 to 30 degrees Celsius - Recommended for surface decalcification if used in 1%
4. Mechanical Hastens decalcification with 70% ROH
agitation
WASHING OUT 5. Size & consistency Larger specimens will slow the rate of a. Von Ebner's
of tissue sample decalcification
- HCl + 36% NaCl
 Removal of excess fixative to improve staining and remove - For teeth and small bones
artifacts - Complete decalcification cannot be determined
Methods of Decalcification through chemical means
for excess formalin, osmic acid, and 1. Acids
1. Tap Water
chromates 2. Chelating Agents 3. Formic Acid
3. Ion Exchange Resin - Only weak acid used as a primary decalcifying agent
2. 50-70% ROH for excess picric acid fixatives and 4. Electrical ionization (electrophoresis) - For routine decalcification of post-mortem research tissues,
Gendre’s Most widely used, stable, easily available,
small pieces of bone and teeth, immunohistochemical
staining
3. Alcoholic for excess mercuric chloride - Better nuclear staining, less tissue distortion than HNO3
iodine relatively inexpensive - Addition of citrate  faster decalcifying rate
1. Nitric acid
2. Hydrochloric acid
3. Formic acid acid
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DAVAO DOCTORS COLLEGE

a. Formic acid-Sodium citrate solution MEASURING EXTENT OF DECALCIFICATION
- P: autopsy, BM, cartilage, research tissues
MEDICAL LABORATORY SCIENCE DEPARTMENT

4. Trichloroacetic acid
Decalcification must be frequently monitored to avoid maceration of
tissue. The methods of measuring extent of decalcification are as
STUDENT NOTES: GPHCT
- Small bone spicules follows:
- Good nuclear staining, does not require washing out
- Weak decalcifying agent and very slow Physical/Mechanical Test DEHYDRATION ACETONE
- Touching and bending tissue using fingers; and poking using - Follows fixation, and decalcification (if applicable)  Fastest dehydrant (1/2 to 2 hours), thus for rapid biopsies
5. Sulfurous acid fine needle or a probe - It is the removal of water from tissue in preparation for  Clear, colorless, and more miscible with epoxy resins than
- Very weak agent thus for minute pieces of bone pieces - Rubbery consistency, and soft = Tissue is decalcified impregnation alcohol
only - DADV: vague and inaccurate artifact production, destruction of - Since most fixatives are aqueous solutions, placing the fixed  DADV: Flammable, extremely volatile, and not recommended
cellular details, small calcified foci may not detected tissue in molten paraffin will not achieve impregnation because for routine work because of considerable shrinkage and
6. Chromic acid (Flemming's Fluid) paraffin wax and water (from fixative) do not mix. Hence, brittleness
- Both a fixative and decalcifying agent Radiologic Method or X-Ray dehydration must be done.  Lipids are removed from tissue
- Minute bone spicules - ADV: most ideal, most sensitive, and most reliable method; can - Done as brief as possible and at a tissue-to-fixative ratio of 1:10
- DADV: highly corrosive to skin, carcinogenic, and detect smallest focus of calcium DIOXANE (DIETHYLENE DIOXIDE)
environmental toxin - Rinse decal agent from tissue  Put tissue on waterproof Characteristics of Ideal Dehydrating Agents  Both a dehydrant and clearing agent
polyethylene sheet on top of X-ray film  Expose to X-ray for 1 1. Rapid action, with minimal tissue shrinkage and distortion  ADV: Less tissue shrinkage, prolongation is possible
7. Citric acid-citrate buffer solution minute at 30kV  Leave until film is developed 2. Should not evaporate fast  DADV: extremely dangerous because of toxicity, and risk of
- Excellent nuclear and cytoplasmic staining but too slow - Opaque result = Incomplete decalcification 3. Able to dehydrate even fatty tissues explosion; expensive, tissues will have poor ribbons
- pH 4.5 - DADV: very expensive, never used with HgCl2-fixed tissues 4. Should not harden tissues excessively  Methods:
(radio-opacity, = xrays do not pass through) 5. Should not remove stains 1. Graupner's - pure dioxane  paraffin
CHELATING AGENTS 6. Non-toxic, and not a fire hazard 2. Weiseberger’s - wrapping tissue in gauze and suspension
Chemical Method to bottle containing dioxane with
- Principle: Use of other salts to form weakly dissociated - Simple, reliable, and convenient Common Dehydrating Agents anhydrous calcium oxide or quicklime
complexes with calcium salts for ease of removal - PCPL: precipitation of calcium hydroxide or calcium oxalate I. Alcohol
- For immunohistochemistry, enzyme staining, and electron - Addition of strong ammonia to the discarded decalcifying fluid II. Acetone  Note: Tissues treated with chromic acid must be thoroughly
microscopy (until solution becomes ALK). III. Dioxane washed with water prior to dioxane treatment.
- Duration: 1-3 weeks for small specimens; 6-8 weeks for longer  Cloudiness = presence of Ca in the discarded fluid IV. Cellosolve
& dense bones  If no cloudiness, add saturated aqueous solution of ammonium V. Triethyl phosphate CELLOSOLVE/ETHYLENE GLYCOL MONOGLYCOL ETHER
- Optimum pH is 7-7.4 oxalate, and let it stand for 30 minutes VI. Tetrahydrofuran  ADV: no shrinkage in prolongation
- Examples: Cal-Ex & Versene (EDTA)  Cloudiness = incomplete decalcification  DADV: Combustible at 110-120OF, toxic to reproductive, fetal,
urinary, and blood systems
ALCOHOLS  If cannot be avoided, propylene-based glycol ethers should be
ION EXCHANGE RESINS POST-DECALCIFICATION  Done in ascending grades to avoid distortion of tissue: used instead of ethylene-based
Removal of decalcifying agent through:
Hastens decalcification by removing calcium ions from formic 70% ROH  90% ROH  100% ROH
- TRIETHYL PHOSPHATE
acid-containing decalcifying solutions, thereby increasing 1. Immersion in saturated lithium carbonate or 5-10% aqueous  For delicate tissues, start at 30%
 Initial alcohol conc. depends on the size and nature of tissue and - A:rapid, little distortion and hardening
solubility from the tissue sodium bicarbonate solution for several hours;
- Ion exchange resin (ammonium form of polystyrene resin) is 2. Rinsing in tap water (small spx=30 mins; large spx=1-4 hours); the fixative used
spread over the bottom of container  put tissue on top  add  Strong initial conc.  shrinkage and brittleness TETRAHYDROFURAN
3. For frozen section, acid decalcified tissue is stored in formol
decalcifying agent (20 to 30 times the volume of the tissue)  Prolonged dehydration in less than 70% ROH  tissue  Both a dehydrant and clearing agent
- Not recommended for fluids with mineral acids such as HNO3 saline with 15% sucrose or PO4-buffered saline (PBS) with 15-
and HCl 20% sucrose at 4OC before freezing maceration  Dissolves fats
- Duration: 1 to 14 days  37OC will hasten dehydration rate (for urgent exams)  Most staining procedures give improved results with THF
TISSUE SOFTENERS  To ensure complete dehydration:  Has rather offensive odor, thus room must be well-ventilated
ELECTROPHORESIS  Add atleast ¼ deep layer of anhydrous Cu2SO4 at bottom
Performed prior to dehydration or sectioning; for unduly hard tissues  Toxic to eyes (conjunctival irritation) and skin (Teflon gloves may
that may damage microtome knives. The agents are as follows: of container, and cover with filter paper be used, but it is recommended to avoid THF use)
- Calcium ions (positively charged) moves to cathode (negative  Bluing of copper sulfate crystals indicates full saturation of
electrode) 1. Perenyi's dehydrating fluids with water, thus alcohol must be changed with
- For small bone fragments ADDITIVES
2. 4% Aqueous Phenol a fresh solution
- Shorter time for calcium removal because of heat and 1. 4% phenol – added to 95% ETOH; softener for hard
3. Molliflex (Effect: tissues appear swollen/soapy [not a
electrolytic reaction involved Ethyl alcohol (Ethanol) tissues
negative effect]) 1.
Uses 88% formic acid Best dehydrant because it is fast-acting, mixes with water 2. Molliflex (glycerol alcohol mixture) - softener
- 4. 2% HCl -
5. 1% NaCl in 70% ROH and many organic solvents, and penetrates tissues easily
References:
- Not poisonous and not very expensive
1. Bruce-Gregorios, J. H. (2016). Histopathologic techniques.
- Clear, colorless, flammable
(2nd Revised Edition). Makati, PH: Katha Publishing
References: 2. Methanol
(611.0182/B83)
1. Bruce-Gregorios, J. H. (2016). Histopathologic techniques. - For blood & tissue films, smears 2. Lo, R., Orillaza, M., Madrid, M., Santiago, F., & Aguilar, P.
(2nd Revised Edition). Makati, PH: Katha Publishing 3. Butanol (2015). Basic histopathologic techniques. Metro Manila, PH:
(611.0182/B83) - Plant and animal microtechniques C & E Publishing
2. Lo, R., Orillaza, M., Madrid, M., Santiago, F., & Aguilar, P. - Less tissue shrinkage and hardening but slow
(2015). Basic histopathologic techniques. Metro Manila, PH:
C & E Publishing
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MEDICAL LABORATORY SCIENCE DEPARTMENT MEDICAL LABORATORY SCIENCE DEPARTMENT
STUDENT NOTES: GPHCT STUDENT NOTES: HPCT
CLEARING  Clove Oil IMPREGNATION
o For skin and smooth muscle, but tends to be  AKA Infiltration Paraffin Wax Substitutes
 Removing dehydrant from tissue, and replacing it with a adulterated  Removal of clearing agent from the tissue and replacing it with 1. Paraplast
fluid miscible to both dehydrant and embedding agent the infiltrating media  Mixture of pure paraffin and synthetic plastic polymer
 Makes tissues “transluscent” or transparent, hence the  Carbon tetrachloride  This infiltrating media will completely fill all tissue cavities, thus (Dimethyl sulfoxide); more elastic and resilient
term clearing o Properties and disadvantages are similar to giving firm consistency, as well as allow easy handling and  MP: 56-57OC
chloroform but is relatively cheaper cutting of thin tissue 2. Embeddol – less brittle, and less compressible (MP: 56-57OC)
 Most are flammable fluids and have low boiling points
 Incomplete Impregnation  Airholes in tissue sections 3. Bioloid – semisynthetic; for embedding of eyes
 Excessive clearing may cause brittleness  Tetrahydrofuran 4. Tissue Mat – has rubber
 Main factors: method of impregnation, nature and size of tissue,
o Shortened turnaround time clearing agent used 5. Ester Wax
Characteristics of a Clearing Agent
 MP: 46-48OC
 Miscible with alcohol, paraffin, and mounting media  Methyl benzoate and Methyl salicylate Types of Tissue Impregnation and Embedding Media  Harder than paraffin thus used with sliding/sledge-type
 Causes minimum shrinkage o For double embedding 1. Paraffin microtome
 Should not dissolve Aniline dyes o Slow acting 2. Celloidin (Colloidin)  Water insoluble but soluble in 95% ethanol, thus prior
 Should not evaporate quickly in water bath 3. Gelatin clearing is not needed
 Makes tissues transparent 4. Plastic  But Cellosolve, and xylene may be used if indicated
OTHERS
 Gum Syrup & Glycerin used when clearing directly from 1. Paraffin 6. Water Soluble Waxes (Polyethylene glycol)
Solutions water (as in a frozen section)
 Xylene/Xylol  Simplest, common and best media for routine processing  MP: 38-42OC or 45-56OC
 Oil of Bergamot for skin and smooth muscle  E.g. Carbowax - most common
o Most commonly used clearing agent  ADV: sections are cut easily without distortion; very rapid
 Oil of Origanum for skin (24hrs); permits many staining procedures; o Hygroscopic = absorbs water; no need for
o 15-30 mins to 1hr working time (most rapid)
o Colorless  Oil of wintergreen artificial oil, for delicate tissues  DADV: not for fatty tissues; must fully impregnate the dehydration and clearing
o Not for nervous tissues and lymph nodes because  Carbon Disulfide for smooth muscle; has foul odor tissue to avoid tissue crumbling o Easily dissolved in water, thus sections are difficult
will cause hardening and shrinkage  Carbol Xylene for friable tissues  Melting point for routine work: 56OC to float out and mount
o Becomes milky when tissues are not completely  Terpineol for delicate tissues like eyes o Never overheat (>60OC) – causes brittleness, [Remedy: add soap to water, or 10% polyethylene
dehydrated  Phenol for smooth muscles shrinkage, hardening; destruction of lymph tissue glycol 900 in water]
 Benzene excellent clearing agent  Paraffin oven – maintain at 2 to 5 OC higher than MP of wax o Neutral fats and lipids can be demonstrated
 High Test Aviation
o Urgent biopsies and routine purposes (15 to 60 Lead-free gasoline  Used pure o Not exposed to too much heat, thus for enzyme
mins); very volatile in paraffin oven o Wax must be filtered first using coarse filter paper histochemistry
o Damages the bone marrow leading to aplastic such as Green’s No. 904 in wax oven at 2OC higher
anemia than MP of wax 2. Celloidin
 Toluene o Reusable only once, but remove water first by boiling  AKA colloidin
o Substitute for xylene or benzene, but slower (1- to 100-105OC  Purified form of nitrocellulose/gun cotton
2hrs)  Specimen with large and hollow cavities which tend to
References: Methods of Paraffin Impregnation collapse; hard and dense tissues; neurologic tissues
o Does not cause brittleness even when tissues are 1. Bruce-Gregorios, J. H.(2016). Histopathologic
left for 1 day; not carcinogenic 1. Manual  Concentration: in 2%, 4%, 8% dissolved in equal parts of
techniques. (2nd Revised Edition). Makati, PH: Katha ether and ROH
o Toxic upon prolonged exposure when used in high Publishing (611.0182/B83)  Atleast 4 changes of paraffin every 15 minutes
conc. 2. Automatic  ADV: Does not require heat for processing; rubbery
2. Lo, R., Orillaza, M., Madrid, M., Santiago, F., &
 Chloroform  Uses machines like Autotechnicon and Elliot Bench-Type  DADV: very slow (days to weeks)
Aguilar, P. (2015). Basic histopathologic techniques.
o For tissue blocks up to 1cm thick; recommended for Metro Manila, PH: C & E Publishing Processor, which fixes, dehydrates, clears, and infiltrates  Methods:
large & tough tissues; CNS tissues, lymph nodes, tissues o Wet Celloidin
and embryos  Infiltration is usually at stations 11 and 12  For bones, brain, teeth
o Toxic to liver, tissues tend to float  ADV: Has constant agitation  speedy procedure  Procedure:
o Does not make tissues translucent  NOTE: Any odor in clearing agent indicates that the 1. Fixation & Dehydration
 Cedarwood oil paraffin wax should be changed 2. Place tissue in ether-alcohol
o For CNS tissues and cytology, smooth muscle, skin  Wax bath thermostat should be set atleast 3 degrees 3. Thin celloidin
o Becomes milky in prolonged storage above the MP of paraffin 4. Medium celloidin
o Forms crystals with acetic-alcohol fixed tses 3. Vacuum Embedding 5. Thick celloidin
(remedy: heat to 200OC prior to using)  Fastest (25-75% reduction of usual impregnation time) 6. Remove specimen and the put it in fresh thick
 Uses embedding oven with negative atmospheric pressure celloidin
 Aniline Oil  rapid removal of air bubbles (e.g. lungs) and clearing 7. Keep in jar or dessicator until ether-alcohol
o P: for insects, embryos, and delicate tses agent rapid infiltration evaporates
8. Store tissue block in 70%-80% alcohol
 For urgent biopsies, delicate tissues (e.g. CNS, eyes)

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 Complete impregnation: No fingerprint marks on o TissueTek – has paraffin reservoir, tissue

surface of tissue block tank, warm plate and cold plate (-5OC)
4. Disposable o Peel-away (thin plastic mold)
DAVAO DOCTORS COLLEGE
o Dry Celloidin molds o Ice tray (inner mold smeared with MEDICAL LABORATORY SCIENCE DEPARTMENT
 For whole eye sections
 Uses Gilson’s mixture (equal parts of chloroform
glycerin or liquid paraffin),
o Paper boats STUDENT NOTES: HPCT
and cedar wood oil) for storage, instead of 70% to
80% alcohol Other Embedding Methods
TRIMMING
o Nitrocellulose Method/Low Viscosity Nitrocellulose 1. Double embedding method– celloidin first, then paraffin; for  Removal of excess wax using knife or cutter after the wax block Types of Microtome
(LVN) large blocks of dense tissues; obsolete is removed from the tissue cassette or paper boat The cutting rate depend upon the type of tissue, the size of the block
 Has lower viscosity, thus can be used in high  Formation of truncated pyramid and exposure of the tissue and the model of the microtome that is used.
concentrations, and rapid tissue penetration 2. Plastic (Resin) embedding surface for ease of sectioning
 ADV: Harder tissue block, thus thinner sections are  For High resolution light microscopy of thinner than usual 1. Rocking or Cambridge  Simplest
 Allow tissue blocks to fit into the block holder of microtome
possible sections, renal biopsies, BM biopsies Inventor: Paldwell Trefall  Cutting section: 10-12µm
 At least 2mm should surround the tissue block
 DADV: Explosive when dry d/t nitrates  For small and large paraffin
o Coarse Trimming
 Plasticizer (e.g. oleum ricini or castor oil) is needed Classes: blocks
o Fine Trimming
to prevent tissue cracking in chrome-mordanted o Epoxy  not for serial sections
tissues  For electron microscopy because of slightly curved
MICROTOMY
 Most widely applied, but carcinogenic due to planes
3. Gelatin vinylcyclohexane dioxide (VCD) component  AKA Sectioning
 Formation of uniformly thin slices/sections/ribbons from the 2. Rotary  Most common
 Rarely used  Types: Inventor: Minot
tissue block with the use of a microtome in order to facilitate  Media: Paraffin
 For histochemical, enzyme studies, and frozen sections  Bisphenol A (Araldite) – slow
studies under the microscope  Excellent for serial sections
 ADV: Water soluble (no dehydration and clearing needed)  Glycerol (Epon) – low viscosity
 Sections usually form ribbons due to slight heat generated 3. Sliding  Media: Celloidin
 DADV: may decay  Cyclohexene Dioxide (Spurr) – very low viscosity;
between the block and the knife edge during the process of Inventor: Adams  Very hard and rough tissue
 TSE must be <2-3mm thick fastest
cutting. blocks
 Procedure:
 Complete ribbons are picked up with camel hair brush, forceps  Types:
1. Wash out of fixative o Polyester
or fingers. o Base-sledge
2. Put tissue in 10% gelatin with 1% phenol  For electron microscopy; seldom used
o Standard sliding – more
3. 20% gelatin with 1% phenol
Microtome dangerous d/t moving
4. Fresh 20% gelatin with 1% phenol o Acrylic Plastics
Principle: Spring-balanced or pawl is brought into contact with, and knife
5. Cool in refrigerator  For High resolution light microscopy
turns a ratchet feed wheel connected to a micrometer screw, which is 4. Freezing  Frozen sections
6. 10% formalin  E.g., polyglycol methacrylate (GMA), methyl metacrylate
in turn rotated, moving the tissue block at a predetermined distance Inventor: Queckett (undehydrated tissues like
 1% Phenol must be added to prevent molds (MMA)
towards the knife for cutting sections at uniform thickness. fat) for rapid diagnosis
 TSE:IMPERGNATING AGENT ratio is set at 1:25  Benzoyl peroxide – catalyst; forms radicals, which are
site for polymerization  Stage for block holder is
 Acrylic plastics must be stored in dark bottles to prevent hollow and perforated,
Microtome Basic Parts attached to a flexible lead
EMBEDDING radical formation and premature polymerization
 Embedding media may be stained, thus use  Block holder/chuck/cassette clamp – where the tissue is held in pipe containing carbon
 AKA Casting, Blocking position dioxide.
 Placing the impregnated tissue into a mold with embedding hydrophobic MMA
 Knife Carrier and Knife – for actual cutting of tissue sections  CO2 as propellant/freezing
media, and then allowing the media to solidify  Pawl, Ratchet Feed Wheel and Adjustment Screws – to line up agent (2 to 3 minutes)
 Orientation: Arrangement of the tissue in a precise position the tissue block in proper position with the knife, and to adjust
in the mold during embedding the proper thickness of the tissue 5. Cryostat/Cold  More common than freezing
References:
 Surface to be cut should be parallel to bottom of the mold Microtome microtome
1. Bruce-Gregorios, J. H. (2016). Histopathologic techniques.
 Molds should bear the accession number  STAT frozen section
(2nd Revised Edition). Makati, PH: Katha Publishing
 Procedure: Care of the Microtome (intraoperative diagnosis)
(611.0182/B83)
o Put tissue with label on a mold, immerse them in melted  Brush away accumulated paraffin and small pieces of  Chamber maintained -5 to -
2. Lo, R., Orillaza, M., Madrid, M., Santiago, F., & Aguilar, P.
paraffin at 5-10 OC higher than MP of wax, then rapidly cool tissues with soft brush after sectioning 30OC with rotary microtome
(2015). Basic histopathologic techniques. Metro Manila, PH:
in a refrigerator at -5 OC, or in cold water  Excess paraffin and tissues may later on interfere inside
C & E Publishing
with the cutting of tissue blocks  Cutting Section: 4 µm
Embedding Molds  Xylene – may also be used for cleaning some
1. Leuckhart’s L-shaped strips of heavy brass and metal; parts of the microtome
Embedding mold reusable and adjustable  Oil movable parts to prevent rusting. 6. Ultrathin  Cutting section: 0.5µm
 Cover microtome to prevent accumulation of dust.  Media: Plastic
2. Compound Interlocking plates resting on flat metal
 For electron microscopy,
embedding unit base; for batch embedding
tissues fixed with osmic acid
3. Plastic Special stainless steel base mold fitted with  Uses special diamond knife
embedding rings a plastic embedding ring (serves as block (ADV: very sharp and no
and base molds holder during cutting) easy dulling)

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Microtome Knives SHARPENING  30 double strokes - given to each side of the knife. • Celloidin sections do not come off in ribbons and have to
Plane-Concave Biconcave Plane-  Badly nicked knives with blunted ends have to undergo sharpening  Advantages: Time-saving; produce well sharpened knives be collected into 70% alcohol immediately.
Wedge in order to ensure optimum sectioning of tissue blocks. with uniform bevels
Length Length: 25mm 120mm 100mm  Sharpening of the knives involves 2 stages, namely:  Disadvantage: Expensive 3. Frozen Sections
1) Honing • Methods of preparing frozen section
Characteristic One side is flat Both sides Both 1) Cold knife procedure
2) Stropping
Other side is concave sides 2) Cryostat procedure (cold microtome)
concave straight Stropping
Honing (NOTE: More on RAPID TISSUE PROCESSING topic)
Embedding Less More Paraffin Paraffin Removal of burrs and polishing of cutting edge
Removal of nicks and irregularities on the knife edges
Medium concave concave Materials:
Materials: FLOTATION/FLOATING-OUT
Celloidin Paraffin  Paddle strop made of horse leather attached to a solid back,
 Hones
in order to prevent sagging.
- Natural sharpening stone or hard grinding surface • For paraffin sections
Microtome Sliding Base- Rotary Base- - Usually dry thus require oiling
- Long enough to allow the whole length of knife edge to be • Sections are floated out on a water bath set at 45-50°c
sledge, sledge - Vegetable oil (e.g. castor oil) applied on the back of the
sharpened in a single stroke (approx. 6-10°C lower than the melting point of the wax used
Rotary horse leather
- Wide enough to sufficiently support and prevent the for embedding the tissue.)
or - Not mineral oil because it tends to blister and destroy
rocking of the knife. • Flotation Bath
Rocking the leather
o Belgium Yellow – most common; best result • 5 to 10OC↓MP of Wax
NOTE:
o Arkansas – has more polishing effect • Inside is specifically colored enamel black
 A good cutting edge must be able to cut good sections from a Procedure: Toe to Heel movement, Edge Last
o Fine Carborundum – coarser; for badly nicked knives • TSEs flatted after 30sec; removes tse
paraffin wax block about 2-3microns thick, without any serration - The procedure is the reverse of honing
o Plate Glass (8x3x1in) – excellent wrinkling
 Too soft cutting edges - likely to become easily dull 1. The knife is fitted with its appropriate knife back
 Lubricants • Dimensions: d=11in, h=4in, 2L capacity
 Too hard cutting edges – likely to produce nicks or jagged edges 2. Knife is laid obliquely on the strop and with the cutting
o Soapy water • Regulated temp. to flatten the sections and prepare them for
edge behind
Angles o Mineral oil mounting into the slides/slider
3. Edge last is pushed backward and drawn forward
o Clove oil • Slides (76x25mm, 1-1.2mm thick, frosted)
1. Bevel Angle: 27° to 32°
o Xylene • Sections should not be left on the water bath for a long
 Angle between the cutting edges Precautions:
o Liquid paraffin time (30 seconds will be enough) to avoid undue expansion
 Maintained by slide-on back (spring-loaded, semi-circular  The knife should always be wiped clean with a “soft” cloth
metal sheet slipped onto the knife)  Knife sharpeners before and after series of stropping (NEVER use paper or and distortion of the tissue.
1. Cutting angle (15°) - angle between the face of a cutting tool and  Flat Glass plate with finely powdered aluminum oxide cloth) • Adhesives may be used for adhesion of tissue to the slide
the surface of the work  May be used for grinding and removing nicks  The knife edge is the oiled or greased to preventing it (more on ADHESIVES topic)
2. Clearing Angle - knife should be inclined at 5-10° from the cutting  Diamantine from rusting • Folds or creases sections – may be removed by stretching the
plane so that the cutting facets (bevel angle) will not compress the  For final polishing  Pressure during the first stropping strokes should be quite sections gently
block during the process of cutting. Procedure: Heel to Toe movement, Edge first light since the natural compressibility of the leather is • Bubbles – may be teased out beneath the sections by means
1. Clean hone with xylene to remove scattered particles of what actually does the work of needle
stones and metal  Speed in stropping should be avoided • Selected sections for staining should be fished out in a vertical
Other Knives and Blades 2. Cover with lubricant  Wax must not be allowed to come in contact with the position
3. Knife is fitted to its corresponding back, placed on one end strop.
1. Disposable o Widely used now because cheaper; of the hone with cutting edge first DRYING THE SLIDES
Blades honing and stropping are no longer 4. With cutting knife edge first, the “heel” (handle end) is drawn
common practice; 40-120 double strokes
obliquely or diagonally towards the operator on the stone  Plane wedge knife: both sides are required for • Mounted sections are placed in a paraffin oven to dry.
o coated with polytetrafluoroethylene (for until the “toe” (head portion) is reached.
ease of ribboning) stropping • 45 – 55°c for:
5. Honing is then continued until all the teeth in the knife edge  Plane – Concave knife: only the concave surface • enzyme digestion
o E.g. Magnetic knives in cryostat have been eradicated” should be stropped. • chemical extraction
6. Washed with water after using to simply remove the metal • metallic impregnation
2. Glass Knives/ o For ultrathin microtomes collected during the process • enzyme localization technique
Ralph Knives 7. After honing, wipe off the oil or soap from the knife with
TYPES OF TISSUE SECTIONS • Hot plates are not recommended because they can cause:
3. Diamond o For resin blocks on ultrathin xylene, then strop it thoroughly……
1. Paraffin (4-6µm)  Overheating
knives microtomes; brittle and expensive
• Successive sections will usually stick edge-to-edge (knife)  Dust falling – onto the section during drying period
10-20 strokes per surface for Minot or Plane-wedge knife • Metal racks with 25-slide divisions are used to store the
4. Safety razor o For partially calcified materials, paraffin due to local pressure with each cutting stroke, thereby
• For plane-concave knives, only the concave surface mounted sections during the drying process which usually
blades and frozen sections. forming a ribbon. (remedy: cut slowly)
should be rubbed on the hone takes 5 minutes in the heated oven. Once dry, the whole rack
o Easily replaced when dull and produces • Sections are removed in ribbons of ten to allow easy
• Plane-wedge & plane-concave is provided with “backs” to of slides can be taken for manual staining.
good tissue sections same as with location of serial sections.
maintain the correct bevel angle (27-32°) throughout
microtome knives honing
o Unsatisfactory for sections less than 2. Celloidin (10-15µm) References:
10micra • The blocks are trimmed in the same manner as in paraffin 1. Bruce-Gregorios, J. H. (2016). Histopathologic techniques.
NOTES:
blocks (2nd Revised Edition). Makati, PH: Katha Publishing
Mechanical Honing
• To avoid dehydration and shrinkage, sections are usually (611.0182/B83)
 Utilizes a machine that make use of vibrating frosted slide
cut by the wet method, both the sections and the block 2. Lo, R., Orillaza, M., Madrid, M., Santiago, F., & Aguilar, P.
plate or wheel driven by electrical motor.
being kept moist with 70% alcohol during cutting. (2015). Basic histopathologic techniques. Metro Manila,
 The knife is pressed against the flat side of a rotating glass
wheel. PH: C & E Publishing
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DAVAO DOCTORS COLLEGE 4. Regressive Staining 6. Counterstaining

 First, the tissue is overstained , and then the excess stain is  application of a different color or stain to provide contrast and
MEDICAL LABORATORY SCIENCE DEPARTMENT removed for specific periods of time or until the desired background
STUDENT NOTES: HPCT intensity of color is obtained
Example:
Differentiation/Decolorization o Cytoplasmic Stains
 selective removal of excess stain from tissue
STAINING Methods of Staining Red Yellow Green
 The process of applying dyes on the sections 1. Simple or Direct staining  Eosin Y   Light Green
 gives color to sections by using simple alcoholic/aqueous dye 3 Classes of Differentiators
 To see and study the architectural pattern of the tissue, the  Eosin B  SF
[Link] Forms a soluble salt with the
physical characteristics of the cells, and the structural solutions   Rose Bengal  Lissamine
Differentiator metal so that the latter is dissolved
relationship of the tissue and their cells Example: methylene blue, eosin
out
 Made possible because of: Varying affinity of tissues and cells Example: HCl; HAc o Nuclear Stains
for dyes 2. Indirect staining
 Nucleus (acidic) attracts basic dyes, cytoplasm (basic) attracts  action of dye is intensified by adding another agent: [Link] Oxidizes the dye to a colorless Red Blue
acidic A. Mordant Differentiator substance  Neutral Red  Methylene Blue
Classification of Staining B. Accentuator Example: K ferricyanide/ K  Safranin O 
permanganate   Celestine Blue
1. Histological Staining Mordant 
 there is direct interaction between the tissue  acts as a link or bridge between the tissue and the dye [Link]
constituents and the dye/staining solution  an integral part of the staining reaction itself, without which, no Differentiator
 differentiates between nucleus and cytoplasm staining could possibly occur
NOTE: 7. Metallic Impregnation
Example:  Stains are not applied; rather, colorless solutions of metallic salts
1. Microanatomic stains - aka histologic stain A. If the primary stain is basic, the decolorizer is acidic , and vice-
versa are used
2. Bacterial stains
B. Alcohol acts as a differentiator for both basic and acidic dye  The tissue reduces the metallic salt solution, producing opaque,
3. Specific tissue stains Tissue-Mordant-Dye Complex
C. Differentiation is also done to restain faded slide usually black deposits on the surface of the tissue or bacteria
 Most valuable metal used: gold and silver
2. Histochemical Staining (Histochemistry)
5. Metachromatic staining  Metallic instruments are avoided when handling the sections
 microscopic localization of a specific tissue  uses specific dyes that differentiate particular substances by Example:
substance through chemical reactions staining them with a color that is different from that of the stain 1. Ammoniacal silver - reduced by argentaffin cells (melanin
Example:
itself and intestinal glands); explosive
1. Potassium alum with hematoxylin in Ehrlich’s
Example:  ALL metachromatic dyes are cationic or basic
2. Iron in Weigert’s Hematoxylin
1. Perl’s prussian blue - hemoglobin  ALL tissue components showing metachromasia are anionic or 8. Vital Staining
3. Tungsten/ Phosphotungstic acid (PTAH)
2. Periodic Acid Schiff - carbohydrates acidic - selective staining of living cell constituent
4. Copper
 Water is necessary because the stain is lost after dehydration - nucleus is not demonstrated; if so, it means that the cell is dead
5. Chromium
3. Immunohistochemical Staining Example: Cresyl blue - for reticulocyte
6. Iodine - not a metallic mordant
a) Intravital staining
 combination of immunologic and histochemical
- injecting the dye into any part of the body of the animal
techniques that allow phenotypic markers to be NOTE: Metachromatic Dyes Example: Lithium, carmine,
detected and demonstrated under the microscope A. Aluminum/alum salts is the most commonly used mordant;  Methyl Violet/Crystal  Methylene Blue India ink - demonstration of uterus in gravid
gives a blue lake Violet  helminthes
 uses antibodies; stain is at Fc portion B. Ferric salt causes blue-black lake  Cresyl blue  Thionine
- monoclonal, polyclonal  Safranin  Azure A, B, C
- fluorescent-labeled b) Supravital staining
Accentuator  Bismarck Brown
- enzyme-labeled - staining the living cells immediately after removal from
does not participate in the staining reaction, but merely accelerates the  the living body
speed of the staining reaction by increasing the staining power and
Example:
selectivity of the stain
1. Neutral red - best vital dye
NOTE: 2. Janus green - for mitochondria
Example:
A. Orthochromic - color shades same to the color of the dye itself 3. Trypan blue - used immediately because toxic to cell
1. Potassium hydroxide in Loeffler’s Methylene blue
2. Phenol in carbol thionine and carbol fuchsin

3. Progressive staining
 staining is carried out in a definite sequence with increasing
concentrations and staining time until the desired intensity of
coloring of the different tissue elements is attained
 No washing , differentiation, decolorization
Example: Any stain as long as no differentiation is done
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STAINS AND STAINING SOLUTIONS OTHER STAINING SOLUTIONS 27. Picric Acid Connective tissue References:
 Stains are prepared from two categories of dyes: 28. Prussian Blue Iron and Circulatory system 1. Bruce-Gregorios, J. H.(2016). Histopathologic techniques.
A. Natural dyes Other Stains 29. Rhodamine B Fixes and stains blood and (2nd Revised Edition). Makati, PH: Katha Publishing
B. Synthetic dyes glandular tissue (611.0182/B83)
1. Acid Fuchsin- Picric Connective tissue 30. Silver Nitrate Spirochetes, reticulum and fiber 2. Lo, R., Orillaza, M., Madrid, M., Santiago, F., & Aguilar, P.
Natural Dye
Acid stains (2015). Basic histopathologic techniques. Metro Manila,
1. Hematoxylin
(Van Gieson’s) 31. Toluidine Blue Nuclear stain for fixed tissues, PH: C & E Publishing
 derived from the heartwood of the Mexican tree Hematoxylin 2. Acridine Orange RNA: red fluorescence Nissl granules or chromophilic
campechianum (originally found in Campeche, Mexico) DNA: green bodies
 most valuable staining reagent Living vs. Dead cells 32. Victoria Blue Neuroglia in frozen sections
 not a stain; but undergoes “ripening ” to produce the active 3. Acridine Red 3B Calcium salts and phosphatase
coloring agent hematin (oxidation product of hematoxylin) activity
4. Alcian Blue Acid mucopolysaccharide Chief Solvent Used for Stains
Two types of ripening - specific for connective tissue and 1. Alcohol
1) Exposure to air & sunlight (slow; 3-4 months) epithelial mucin
2. Phenol
2) Chemical oxidation (instantaneous) 5. Aniline Blue Counterstaining of epithelial
3. Aniline Water
 tissues
4. Water - must always be distilled unless stated
 6. Basic Fuchsin Acid fast organisms, mitochondria,
 mucin and elastic tissue
 a. Carbol fuchsin
b. Coleman’s Fuelgen Other Materials Used in Staining
 Sodium Iodate Coplin jar Holds 5-9 slides
c. Schiff’s
2. Cochineal Dye
d. Mallory’s fuchsin Slotted staining dishes Hold 5-19 slides
 old histologic dye e. Aldehyde fuchsin Metal/glass staining racks/ Hold 10-30 slides
 extracted from the female cochineal bug Coccus cacti carriers
 widely used as a powerful chromatin and nuclear stain 7. Benzidine Hemoglobin
 Carmine - treated with alum
8. Bismarck Brown Contrast stain for Gram’s Acid
 Picrocarmine - carmine + picric acid (neuropathology) Staining of Paraffin Sections
Fast and Papanicolau
 Best’s carmine stain - carmine + aluminum chloride  “Sections to water”
(glycogen) For Diptheria organisms  Reversing the tse processing prior to using water-miscible
3. Orcein 9. Brilliant Cresyl blue Reticulocytes stains
10. Carmine Fresh material in smear  Deparaffinization – immersion in xylene to remove paraffin
 Vegetable dye; extracted from certain lichens
preparations  “Sections to Alcohol” – deparaffinization + immersion to
 mainly used for staining elastic fibers descending grades of ROH
 initially colorless, but produces blue/violet color when 11. Mayer’s Carmalum
Soln  Clearing may be prolonged
treated with ammonia and exposure to air  Dehydration may remove stains
12. Celestine Blue Routine staining of fixed tissue
NOTE: Litmus, also obtained from lichens has poor staining 13. Axis cylinders of embryos Staining of Celloidin Sections
capacity and is used mainly as an indicator 14. Crystal Violet Amyloid staining and platelets
 Celloidin must never be used with absolute alcohol
15. Giemsa Stain Leukocyte differentiation
16. Gold Sublimate
Synthetic Dye 17. Iodine Oldest stain Restaining Proedures
AKA Coal tar dyes - derived from coal tar Starch granules, amyloid, 1. Immersion in xylene for 24 hrs or gentle heating of samples until
Aniline dyes -derived from hydrocarbon benzene cellulose, carotene and glycogen mounting media is already removed (begins to bubble)
18. Janus Green B Mitochondria 2. Remove coverslip
Chemical Basis of Color in Dyes 19. Malachite Green Ascaris eggs, erythrocytes and 3. Xylene again for 30mins
1. Chromophore bacterial spores stain 4. Tap Water
 Color-bearers 20. Methyl Green Chromatin 5. 0.5% potassium permanganate solution for 5-10mins.
 Causes the color of the dye to appear 21. Methylene Blue Plasma cells, fresh sputum milk 6. Rinse in tap water
2. Chromogen bacterial stain, differentiation of 7. 5% oxalic acid for 5 minutes or until the section is decolorized.
 Color-generating colon and aerogenes bacteria and 8. Rinse in tap water for 5mins
 Chromophores that are bond w/ benzene (C6H6) diphtheria diagnosis 9. Restain
 Composed of an acid & a base (salt-forming 22. Methylene Violet
23. Neutral Red Cell granules and vacuoles of Stains on skin
properties) to become a dye
3. Auxochrome phagocytic cells  Remove by using 0.5% acid alcohol, then rinse with tap water
 Color-increasers 24. Night Blue Acid fast staining
 Responsible for dyeing property of the dye (shade) 25. Orcein Elastic fibers, dermatological
groups studies for delicate and finest skin
fibers
26. Osmium Tetroxide Fat stain

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DAVAO DOCTORS COLLEGE  Difficult due to inaccessibility and presence of gastric fluid
ADHESION  Examine ASAP
MEDICAL LABORATORY SCIENCE DEPARTMENT  For adequate adhesion of smeared material.  8 hours fasting before gastric washing; even water is
STUDENT NOTES: HPCT  Albumin not recommended because it will retain the OG of
Pap stain
prohibited

 Specimens That Require Adhesives : VI. Peritoneal, Pleural & Pericardial Smears
 Urinary sediment  Collection: same as bronchial specimens
CYTOLOGY  (spray fixatives: 12 inches or 1 foot away)
 BAL  Increased fluid indicates pathologic Process
 Avoid vibrations in the container to prevent the cells from
 Histology of exfoliated, abraded or desquamated cells.  Specimens with proteolytic enzymes  Presence of malignant cells usually indicate metastasis
being dislodged
 Concentrated sputum  Add 300 units of heparin per 100 mL of aspirate to avoid
 Optimal time: 1 hour to allow dehydration, adhesion and
 Characteristics of Adhesives: clots
maximal penetration of the fixative
IMPRINT/ABRADED CYTOLOGY  permeable to both fixative & stain
 Examples of Good Adhesives : VII. Urine
 Study of cells directly taken from surfaces of excised specimens NOTE:
 Pooled Serum / Plasma  Collection:
by touching them to a clean glass slide Best (yet flammable) fixative - 95% ethanol-ether
 Celloidin Ether Alcohol  Male - Second voided urine
Routinely used fixative - 95% ethanol
 Leuconostoc Culture  Female - catheterized
 Atleast 50 mL is needed
EXFOLIATIVE CYTOLOGY  If smears cannot be made immediately, place the
 For more reliable evaluation, urine may be collectd and
collected material in 50% alcohol (for all types of
 Microscopic study of desquamated cells from epithelial surfaces. Non-Gynecologic Specimens examined twice: early morning and later in the day
effusions) ---> replaced by Saccamono’s preservative
 Study of cells that have been shed or physically removed  Use of preservative is not recommended
Components: 50% ethanol , 2% carbowax
Respiratory Specimens  For diagnosis of urothelial malignancy and not for
PURPOSE 1. Sputum prostatic carcinomas
 Storage: add ROH & refrigerate.
 Assessing cancerous conditions (staging)  50% - pleural / peritoneal  Demonstrate abnormal cells early in disease
 Detection of asymptomatic cancers  70% - sputum  Fresh, unfixed, atleast 3 consecutive early morning
 Assessment of female hormonal activity (sterility & sputum VIII. Breast Secretions
 95% - urine, bronchial, gastric
endocrine disorders) - maturity index  Patients unable to produce sputum: INDUCED SPUTUM  Collection: Spontaneous nipple discharge
 Determination of genetic sex - Barr bodies by inhalation of aerosol solution for 20 minutes.  Materials: cotton swab or touch prep
 Mailed Specimen: air-dry for 10-15 minutes after 2 hrs
(conglomeration of chromatin, XX chromosome)  Wide mouthed bottle w/ Saccomano’s fluid  For hormonal imbalance testing
fixation & place in a container
 Detect presence of possible infection.  After collection:  Extremely low diagnostic yield for breast carcinoma
1. Pour specimen in petri dish, examine for solid or diagnosis
 If specimen is more than a few drops:
SPECIMENS blood flecked particles  If bloody, benign intraductal papilloma
1. Centrifuge at 2,000 rpm for 2 minutes.
1. Cervicovaginal smears (Pap’s smear) 2. Decant the supernatant, and smear the sediment 2. Remove particles, place on slide, crush w/ another
2. Prostatic & Breast Secretions slide. Evenly distribute. IX. CSF
directly to glass slide, or cytocentrifuge directly
3. Pleural & Peritoneal Fluids 3. Fix for a minimum of 1 hour.  Minimum of 1 mL is necessary
on slide with albumin.
4. Sputum 3. Extra sediment can be used for cell block  Absence of histiocytes & alveolar macrophages indicate
5. Gastric & Bronchial Secretions that only saliva was collected X. Prostatic Secretion
technique.
6. Urine sediments – First morning?  3 Specimen:
a) First morning is not recommended for cytologic II. Bronchial Brushing  Voided urine before massage.
General Processing
evaluation because the cells may degenerate after  Directly smear on two slides using Pull-apart technique  smear of prostatic secretion by massage.
 Viscous Specimen Immerse in Ether-Alcohol ASAP.
spending extended time period in acid environment  Fix immediately to avoid production of Air drying artifacts  Urine after massage
(cervical, vaginal,
in the bladder prostrate  Cytologic examination of 3rd spxn after unproductive
7. CSF secretion): III. Bronchoalveolar Lavage/Bronchial Washing massage is recommended.
  Performed in patients with AIDS to rule out Pneumocyctis
PREPARATION  Mucoid Specimens Smeared thinly then dry smear edges jirovecii infection
 Material from fluids of the body may be examined either (sputum, bronshial before fixing (to avoid runoff) Gynecologic Specimens
by: and stomach IV. Bronchial Aspirate
 Preparation of Smears secretons):  Collection: aspiration (with glass suction apparatus) or I. Vaginal & Cervical Smears
 Preparation of Tissue Blocks (Cell Blocks)  Bloody Specimens: RBC can be destroyed by adding 2- washing (with saline)  Screening test for precancerous condition
5mL of concentrated acetic acid per  Show evidences of malignancy in advanced stage of the  Not diagnostic
100mL of the specimen. disease.  Collection: aspiration of posterior fornix, swabbing
Smears
 Make smears from fresh and moist material on clean slide;  Centri specimen for 20-30 minutes, medium speed  Instruments:
 Watery Specimens Specimen is centrifuged first and the  smear must contain epithelial cells (ciliated bronchial cells  8-inch glass pipette & rubber bulb (for cancer
should not have clumps (urine, exudate, sediment is smeared in an albumin-
 Diamond pen is used to put identifiers on the smear ) + RBC & WBC. cytology)
aspirate): coated glass slide. In the event that
(name, age, date, and type of smear); frosted-end slides  If spxn is scanty, prepare smear in OR to prevent drying.  sterile tongue depressor (for hormonal studies)
there is excess sediment, it may be
may also be used.  Ayre’s spatula
subjected to biopsy.
 Immerse smears immediately in fixative by a single Gastrointestinal Specimens  Cervix brush
uninterrupted motion (exfoliated cells decompose rapidly) V. Gastric lavage, Gastric brush, Submucosal lesion FNA  Precautions for Vaginal Smear Prep:
 Collection: Irrigation &Aspiration  patient must avoid vaginal exam’n or douching 24-48
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hrs before collection. Other Microscopic Examination of Cytologic Smears  Double walled boundary with deep blue stain at the Quantitative Evaluation for Vaginal Cytology
 spread smear thinly in a rotary motion 1. Acridine Orange Fluorescent Technique cytoplasmic periphery 1. Maturation Index (MI) The percentage of cells from the
 glass pipette must be absolutely dry a) Binds DNA --> green/yellow  eccentric nucleus ( glycogen) main layers of vaginal epithelium
 no lubricant or powder must be used i. If increased: Malignancy (superficial, intermediate and deep)
b) Binds RNA --> brick-orange red III. Parabasal Cells Criterion: Pyknosis
Cytologic Collection and Preparation i. If increased: Growth  Smaller, thick, round to oval 2. Acidophilic index (AI) The percentage of cells staining
Endocervical brush - samples of endocervial canal  strongly basophilic cytoplasm pink-orange to red with Pap’s
Vaginal scrape - patients with hysterectomy  larger vesicular nucleus than intermediate cells smear. This is not a reliable index
2. Phase Contrast Microscopy
due to possible
Lateral vaginal scrape - for hormonal evaluation a) The second best choice afte rPap’s staining  2 weeks of age to puberty, after childbirth, abortions & after
pseudoacidophilia.
Four Quadrant Vaginal - for localization of vaginal b) Used for hormonal evaluation of gynecologic specimen menopause
3. Pyknotic index (PI) The percentage of cells with
Scrape adenosis and for cancer detection shrunken, dark and small
Vuvlar scrape - for detection of herpetic lesions IV. Basal Cells structureless nuclei
or carcinoma 3. Interference Microscopy  small, round to slightly oval cells
a) Determines the dry weight of individual cells or cellular  large nuclei (> half of cell vol.)
constituents  strongly basophilic cytoplasm
Papanicolau Staining Method b) Very expensive and complex  found before puberty & after menopause References:
1. Bruce-Gregorios, J. H.(2016). Histopathologic techniques.
 Staining method of choice V. Endometrial Cells (2nd Revised Edition). Makati, PH: Katha Publishing
 Developed by Dr. George Papanicolau (1940’s) VAGINAL HORMONAL CYTOLOGY  like parabasal cells; slightly cylindrical (611.0182/B83)
 Detect human uterine & cervical CA.  less basophilic cytoplasm
 Cytologic evaluation of other secretions  Inexpensive  during & 1-4 days after menstruation
 Not commonly used nowadays
 Transparent blue staining of cytoplasm  performed regularly even in pregnant women without undue risk VI. Endocervical glandular cells
 Excellent nuclear detail is produced  No prior vaginal examination or douching in the last 24 hours  Occur in large group or sheets
 Color range is predictable  upper lateral 3rd of vaginal wall (less contamination &  Honeycomb appearance
 valuable in comparing cellular appearances in smears accessible)  Finely vacuolated pale blue/gray cytoplasm
 Wooden spatula is recommended only in vaginal hormonal
studies VII. Doderlein Bacilli
Papanicolau Stain  LPO:  L. acidophilus
 Makes use of 3 stains:  assess smear & staining quality  most common normal vaginal flora
Stain Cells Stained  detect RBC & WBC  blue to lavender w/ Pap's
 Hematoxylin Nuclear structure  detect type of exfoliated cells  Increased number indicates healthy vagina
 rough assessment of cell proportion  Numerous in: last trimester of pregnancy, infection,
 OG6 (orange green) Cytoplasm of mature cell  HPO: quantitate estrogen deficiency, DM
 Decreased number leads to increase in population of
invasive Lepthothrix species; comes with presence of T.
 Eosin Azure Cytoplasm of immature cell Cells Found in Vaginal Smears vaginalis

Components:
I. Mature Superficial Cells
 Eosin Ferning Phenomenon
 Dark pyknotic nuclei (< 6 u )
 PTA
 True acidophilia (due to estrogen )
 Light Green Stain (36,  "fern"/palm-leaf pattern of salt crystals
 Pseudoacidophilia :
50, 65)  seen on dried cervical mucus
 Vaginal prolapse and drying
 Lithium carbonate  signifies persistent estrogen effect
 Drying of smears
 Bismarck brown  basis for diagnosing early pregnancy
 Infection
 Chemicals

II. Intermediate Cells Criteria for the cytologic diagnosis of normal pregnancy:
Modified Pap’s
 medium sized polyhedral or elongated cells
 It omitted the Bismarck brown dye from the EA solution 1. Marked progesterone effect
 basophilic cytoplasm with vacuoles
 Sharpness of color and brilliant staining reactions are improved
A.) NAVICULAR CELLS 2. At least 50 % of intermediate cells in cluster
 boat-shaped; folds / curls on edges 3. At least some typical pregnancy cells are present
 estrogen-progesterone effect 4. Less than 20 % mature superficial cells
 latter half of menstrual cycle, pregnancy & 5. Doderlein-filled dirty background
menopause.
B.) PREGNANCY CELLS
 round, oval cells
 Translucent basophilic cytoplasm at the center of cell
JALN2020 JALN2020

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CHAPTER 15 ADHESIVES

ADHESIVES AND MOUNTING MEDIA 1. Mayer's Egg Albumin

FORMULA DESCRIPTION
Mounting is the last step in tissue processing that results in a permanent histological preparation suitable for  Egg White 50 cc. Mayer's egg albumin is the most commonly used because it
microscopy, after adhesion of the sections on to the slide and appropriate staining of the tissue. After cutting, sections are  Glycerin 50 cc. is very easy to make, is convenient, and is relatively
floated out on a water-bath. Bubbles accumulating under the ribbon may be removed with a smooth teasing needle, care  Filter and add about 100 mg. crystals of thymol to inexpensive. A drop of Mayer's egg albumin is usually
being taken not to tear the section. Bubbles may also be removed by pulling the ribbon very gently across the long edge of prevent the growth of molds. smeared into the clean glass slide before sections are
a glass slide held below the section in the water bath. When the sections chosen have flattened out, the numbered slide is oriented. Sections which have been creased on cutting may
immersed in the water bath and the section is fished out and drained. After draining, the sections are fixed to the slides. be stretched by gentle heating before attaching them into
slides. During staining, the excess of albumin may also take
This can be done either by leaving the slides in a 37°C incubator overnight, by placing the slides in a wax oven at 56° to
up the stain and interfere with diagnosis; hence, it should be
60°C for 2 hours, or by drying the slides on a hot plate at 45° to 55°C for 30 to 45 minutes. For more delicate tissues like
wiped off from the slide to remove any excessive solution.
the CNS tissue or brain, a longer drying time at lower temperature (e.g. 37°C for 24 hours or longer) is recommended to
avoid splitting and cracking of the section due to excess heat. Another alternative to drying is by the use of adhesives. To For celloidin sections, egg albumin is smeared on the slide.
promote adhesion of sections, adhesives may be spread thinly and evenly on a clean grease-free slide which is then gently The section is then transferred from 95% alcohol bath to the
approximated to the end of the ribbon, and drawn upwards in a near vertical motion. slide, pressed flat on the slide with a smooth filter paper
coated with thin celloidin mixture.
ADHESIVES

An adhesive is a substance which can be smeared on to the slides so that the sections stick well to the slides. The 2. Dried Albumin
choice of slide and adhesive will be influenced by the staining methods to be subsequently applied. Slides must always be FORMULA DESCRIPTION
grease- and dust- free and stored and handled correctly. If staining is to include antigen retrieval (IHC), enzyme pretreatment  Dried albumin 5 gm Dried, and stored in 70% alcohol until it is ready for
for in-situ hybridization (ISH), or prolonged incubation steps, charged slides or an adhesive must be used. Some special  Sodium chloride 5 gm staining.
stains, particularly those that employ alkaline reagents, can also cause sections to lift. Slides must always be accurately  Dissolve in 100 cc. of Distilled Water and add
and appropriately labelled in a manner compliant with local regulations. crystals of thymol.

Adhesives are not necessary for routine staining, provided that the slides are clean and free from grease. However, they
are essential for methods that require exposure of sections to acids and alkalis (especially ammoniacal silver solutions) 3. Gelatin (1%)
during staining. In such cases, the amount of adhesives applied on the slide should be kept to a minimum, since they are FORMULA DESCRIPTION
 Gelatin 1 gm Adding up to 30 ml of 1% aqueous gelatin to the water in a
prone to contamination and bacterial growth that might be confused with real tissue organisms demonstrated by Gram and
 Distilled water 100 ml floating out bath and mixing it well is a most convenient
PAS stains
 Glycerol 15 ml alternative to direct coating of slides.
If clean grease-free slides are used and sections are adequately dried, the sections will not float off during staining and  Phenol Crystal 2 gm
adhesive will not be necessary. There are still certain instances when sections may float from the slide:

For urgent cryostat sections to be submitted for For tissues which have been decalcified 4. Gelatin-formaldehyde mixture
FORMULA
immunocytochemistry
 1% gelatin 5 ml
For central nervous system tissues When sections are to be subjected to high temperatures
 2% formaldehyde 5 ml
For tissues containing blood clot
 Coat the slides with the above mixture. Allow coated slides to dry at 37°C for one hour or overnight before use.

The most commonly use adhesive is Albumin. Albumin solution is prepared by mixing equal parts of glycerin, 5. Poly-L-Lysin e
distilled water and white of eggs, then filtered through coarse filter paper and a crystal of Thymol is added. One disadvantage DESCRIPTION
of using albumin is that it retains some of the stain and gives a dirty background. Thymol resistant organisms growing in the  This aqueous detergent can be purchased as a 0.1% solution which is further diluted 1:10 with distilled water (final
adhesive have been known to contaminate gram-stained sections and cause confusion during microscopic examination. dilution to 0.01%) prior to use. Sections are coated with this diluted poly-L-lysine and allowed to dry. This is widely
used as a section adhesive in immunohistochemistry. PolyL-lysine coated slides must be used within a few days
Poly-L-lysine, also a favorite adhesive, can be bought as a 0.1 % solution and further diluted (1 in 10 with distilled after they are prepared, since its effectiveness as an adhesive slowly decreases in time.
water) when ready to use. Sections are coated with this dilute poly-L-lysine and allowed to dry. With time, the adhesive
ability of this substance slowly loses its effectiveness. Therefore the coated slides should be used within a few days.
6. APES (3-aminopropylthriethoxysilane)
Aminopropyltriethoxysilane (APES) is a better section adhesive and coated slides can be stored for a long time.
Slides are dipped in 2% APES in acetone drained then dipped in acetone, drained again and finally dipped in distilled water. DESCRIPTION
 APES-coated slides are very useful in cytology, particularly for cytospin preparations of proteinaceous or bloody
It is invaluable in cytology particularly for cytosine preparation of proteinaceous or bloody material. material. The slides are dipped in 2% APES in acetone, drained, dipped in acetone, drained again, and finally dipped
in distilled water. They are then placed upright in a rack and allowed to dry. APES-coated slides are better than poly-
L-lysine coated slides because they can be stored for a long time without losing their adhesiveness.

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MOUNTING MEDIUM to the slide for proper identification, while also avoiding any damage to the sections caused by wiping the "wrong" side of
the slide.
If an unmounted stained section is seen in the microscope, differences in light refraction between the glass slides
and the tissue components, may lead to difference in length dispersion; hence, very little microscopic detail can usually be Mounting media may be divided into two main groups:
appreciated. Tissues should therefore be impregnated with a transparent medium that has an index of refraction close to
a. Aqueous Media
that of the glass and the tissue. The mounting medium bonds specimen, slide and coverslip together with a clear durable
film. b. Resinous Media
A mounting medium is usually a syrupy fluid applied between the section and the coverslip after staining, setting
the section firmly, preventing the movement of the coverslip. It protects the stained section from getting scratched, to
facilitate easy handling and storage of the slides, and to prevent bleaching or deterioration due to oxidation, thereby A. AQUEOUS MOUNTING MEDIA
preserving the slides for permanent keeping. The mounting medium also helps prevent the distortion of image during
microscopic examination. Aqueous mounting medium are used for mounting sections from distilled water when the stains would be
decolorized or removed by alcohol and xylene as would be the case with most of the fat stains (Sudan methods) or
Mounting media are often chosen for a specific refractive index (R.I.), which can enhance specimen details or make for metachromatic staining of amyloid. They are usually made up of gelatin, glycerin jelly or gum arabic (to solidify
them invisible. Materials like glass become totally invisible if immersed in a solution of the same refractive index. Refractive the medium), glycerol (to prevent cracking and drying of the preparation), sugar (to increase the refractive-index),
index is important because it governs the contrast between the cellular detail and the background, and also the transparency and a preservative solution.
of the observed sample against the bright field of the microscope. The mounting media must always have an RI higher than
the mounted sample to impart more transparency. Following are examples of common aqueous mounting media:
1. WATER Has a low refractive index, is moderately transparent and evaporates easily, hence is good
The slide carrying the section to be mounted is taken from the last xylene bath with the forceps. Excess xylene is
only for temporary mounting. Also, water does not allow tissues to be examined under the
wiped off from the back of the slide and from around the section. A drop of mounting medium is placed down the center of oil immersion lens. In a wet mount, the specimen is suspended in a drop of liquid (usually
the slide. A clean, dry cover glass is placed on the edge of the slide and gradually inclined downward until it touches the water) located between slide and cover glass.
mounting medium and gently pressed on to the slide while the mounting medium quickly spreads through the whole area
of the section. The slide may then be incubated at 37°C for 12-24 hours after mounting, to harden the medium. The water refractive index of the water improves the image quality and also supports the
specimen. In contrast to permanently mounted slides, wet mounts cannot be stored over
Do not use immersion oil on an uncovered slide. If in a hurry to view a specimen under oil immersion, mount the extended time periods, as the water evaporates.
slip and handle the mount carefully, keeping it horizontal. Oil can be applied, but do not attempt to wipe it off until the
mounting medium is cured - at least overnight or an hour on a hotplate or in an oven. Do not store mounted slides vertically 2. GLYCERIN May also be used as a preservative. It has a high index of refraction and provides greater
for 2 days if cured at room temperature. visibility if slightly diluted with water (for moist sections). This is a very suitable semi-
permanent mounting medium with a refractive index of 1.46, sets quite hard, and will keep
Excessive mounting medium will cause it to ooze out of the sides of the cover glass, and should be carefully wiped sections mounted for years, especially if sealed on the edges with paraffin wax. It is miscible
with a fine cloth moistened with xylene. Excessive blotting, on the other hand, will dry up the section, causing shrinkage and with water, is inexpensive, and is non-poisonous. It is also not necessary to treat the
cracking of the specimen. If the section has to be remounted, the cover glass may be removed by soaking in xylene. Excess specimens with alcohol or organic solvents, which may introduce artifacts and remove
xylene, if not removed, will mix with the mountant and form bubbles on the slide. Too little mounting medium may also cause pigments. This is usually regarded as the standard mountant for fat stains.
improper setting of the coverslip or formation of bubbles on the section, which can be teased out by gently pressing on the
cover glass with a pointed forceps or mounting needle. Setting may be hastened in a hot oven at 50°C for 2 hours. The disadvantage is, that it is difficult to prepare slides that are truly permanent in nature.
Because it is a thick liquid, it can slowly run off a slide that is tilted. A proper sealing of the
CHARACTERISTIC OF A GOOD MOUNTING MEDIUM cover slip corners is absolutely necessary if one wants to store the slides over extended
It should be colorless and transparent. It should not cause shrinkage and distortion of tissues. periods. Do not stack slides for long as the pressure will squeeze glycerin from the mounts.
Glycerin will eventually evaporate and air will penetrate under the cover slip. Glycerin can
It should be freely miscible with xylene and toluene. It should not leach out any stain or affect staining.
be attacked by microorganisms, so one can optionally add a crystal of thymol to avoid
It should not dry to a non-stick consistency and harden It should not change in color or pH. bacteria and fungi.
relatively quickly.
It should protect the section from physical damage and It should be compatible with the adhesive in use As with other solvents it is used because it is cheap, safe and quick to use with little
chemical activity (oxidation and changes in pH). preparation. Ringing the coverslip with a hydrophobic seal will extend the life of mounted
It should be resistant to contamination (particularly It should set without crystallizing, cracking or shrinking (or sections, although cationic dyes will diffuse into the medium over time. Phosphate buffered
microorganism growth). otherwise deform the tissue being mounted) and not react glycerol (RI = 1.47) is commonly used to mount sections for immunofluorescence and
with, leach or induce fading in stains and reaction products glycerol may be added to other agents to retard drying and cracking.
(including those from enzyme histochemical, hybridization,
3. GLYCERIN JELLY FORMULA: Gelatin 10 gm. Glycerol 70 ml. Distilled water 60 ml. Phenol crystals
and immunohistochemical procedures).
(KAISER'S 1880) (preservative) 0.25 gm.
(Refractive Index
1.47) Gelatin is added to distilled water and incubated in a water bath at 60°C until dissolved.
A mounting medium should be chosen that will not fade the particular stains used; for example, basic aniline dyes
Glycerol and then phenol crystals are added and mixed. The solution is labeled, and stored
should be mounted in non-acid containing mountants. Preparations showing the Prussian blue reaction should be mounted in a refrigerator at 40°C. For use, the gelatin must be heated in a water bath or incubator at
in non-reducing media. The mounting medium is usually dispensed from the stock bottles into screw cap collapsible tin 60°C to melt. Too much gelatin makes the jelly difficult to melt and included bubbles found
tubes. This will keep the mountant clean and prevent the concentration and thickening of the solution subsequent to on the slide will not burst. The melted medium should not be shaken or stirred before use,
evaporation. if formation of air bubbles is to be avoided.

Slides should be properly labeled with an identifying case number on the side of the mounted coverslip to. As a Glycerin jelly is the standard mounting medium used when dehydration and clearing with
general rule, a paper label bearing the patient's name, section number and preferably the staining method used, is attached xylene cannot be made (as in fat stains). Pure glycerin has the highest index of refraction

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and thus provides the best viewing and may be optimal for critical or irreplaceable material, B. RESINOUS MOUNTING MEDIA
because old material, when glycerin is mostly evaporated, is easily retrieved with hot water
or steam. The disadvantage is that it should be melted before use (due to the presence of
gelatin). Stains mounted on glycerin jelly tend to fade. Polyvinyl alcohol, often used as a Sections of tissue embedded in plastic compounds (such as epoxy resins) can be successfully mounted in liquid
mountant in immunofluorescence microscopy, has been recommended as an alternative resin of the same type. Sections should be completely dry before applying mountant, which is best set using the
for glycerine jelly. The mountant is not set in the desired amount of hardness and therefore same conditions prescribed for tissue blocks. Resinous media are used for preparations that have been dehydrated
requires "ringing". and cleared in xylene or toluene, and are recommended for majority of staining methods. They may be divided into
natural and synthetic resins. The most important synthetic resins are used for embedding undecalcified bones, and
4. FARRANT'S FORMULA: Gum arabic 50 gm. Distilled water 50 ml. Glycerol 50 ml. Sodium merthiolate for electron microscopy.
MEDIUM 0.025 gm Dissolve gum arabic in distilled water with gentle heating and add glycerol and
(Refractive Index 1.43) sodium merthiolate. Mix well and label. Following are examples of resinous mounting media:
This gum arabic medium does not solidify upon storage and therefore does not need to be
1. Canada Balsam Canada Balsam is a natural resin extracted from the Canadian tree, Abus Balsamea, usually
heated before use. However, it takes a longer time to harden and may therefore require
dissolved in xylene in an incubator at 37°C or paraffin oven at 58 °C, and filtered, obtaining the
ringing. (Refractive Index 1.524)
desired consistency by controlled evaporation of the solvent. The solution can be made neutral
or acid by adding excess amounts of calcium carbonate or salicylic acid, allowing the mixture
Arsenic trioxide may be used as a substitute of sodium merthiolate for preservation of the
to settle, decanting the supernatant liquid into a stock bottle, and discarding the residue.
medium. Addition of 50 gm. potassium acetate will produce a neutral (pH 7.2) instead of an
acid (pH 4.4) medium, and therefore, will raise the refractive index to 1.44.
It is a transparent, almost colorless oleoresin that adheres firmly to glass and sets to a hard
consistency without granulation. However, it darkens slightly with age and slowly becomes acid
because it oxidizes xylene, thereby causing gradual fading of many stains.
5. APATHY'S MEDIUM FORMULA: Pure gum arabic (crystals not powder) 50 gm. Pure cane sugar or sucrose 50
The harmful solvents which, constitute a health hazard such as xylene, may limit the use of
(Refractive Index 1.52) gm. Distilled water 50 ml. Thymol crystals 0.05 gm.
Canada Balsam as a mounting medium. The use of nontoxic solvents like histomount instead
of xylene like histomount may be less of a health hazard but may cause other problems such
This medium is used for methylene blue-stained nerve preparations and as a general
as slow hardening and premature darkening. Benzene may be substituted for xylene as a
purpose aqueous mountant. It is one of the most useful aqueous mountants for fluorescent
solvent. The medium can only be neutralized temporarily since the mixture becomes acidic and
microscopy, being virtually nonfluorescent. Von Apathy’s medium is not compatible with
changes into a brown color upon storage. Calcium carbonate chips may be added to maintain
normal histological stains. The pH of the medium is near 4.0 (highly acidic) so stains fade
its neutral reaction. The solution acidifies and darkens with age and upon exposure to sunlight,
or bleed into the medium. Addition of 50 grams potassium acetate, 20 grams of calcium
for which reason it should be kept in a dark glass bottle. Stains are usually not preserved due
chloride or 10 grams sodium chloride can raise the pH to near 7.0 and will prevent
to acidity on prolonged exposure.
"bleeding" of metachromatic stains for amyloid. The medium sets quite hard, has a higher
refractive index, and does not require ringing.
Canada balsam is recommended for whole mounts and for thick sections because it does not
shrink much. It sets hard without granulation; it is, however, quite expensive. As Canada
6. BRUN'S FLUID FORMULA: Glucose 24 gm. Glycerine 6 ml. Spirits of camphor 6 ml. Distilled water 84 ml.
Balsam does not mix with water, mounting in it implies the use of a sequence of dehydration,
Mix, shake well, and filter. Store the solution in a well-stoppered bottle.
starting with low grade alcohols, followed by high grade alcohols, absolute alcohol, mixed
clearing agents plus alcohol, clearing agents, clearing agents mixed with xylene, pure xylene,
Brun's Fluid is recommended for mounting frozen sections from water. Frozen sections that
and balsam dissolved in xylene. Toluene or benzene could be used instead of xylene.
are mounted directly from water or paraffin sections which require dehydration and clearing,
usually should be mounted on glycerin, gum syrup or Brun's fluid.
2. DPX This is a resinous medium recommended for small tissue sections but not for whole
(Dibutyl Phthalate and Xylene) mounts because of shrinkage produced on drying; hence, it should be used in excess
(Refractive Index 1.532) amounts. It is a colorless, neutral medium in which most standard stains are well
preserved. It is prepared by dissolving the common plastic, polystyrene, in a suitable
hydrocarbon solvent (usually xylene).

It tends to set quickly and, in doing so, often retract from the edge of the coverslip. It
has a greater advantage over Canada balsam in that slides can be cleaned of excess
mountant simply by stripping it off after cutting around the edge of coverslip.

3. XAM Xam is a synthetic resin mixture in xylene, available in a pale yellow or colorless solution. It
dries quickly without retraction, and preserves stains well. Sections are quickly mounted from
(Refractive Index 1.52) xylene.

4. CLARITE Clarite (or Clarite X) is a synthetic resin which is soluble in xylene (it is used as a 60% solution
in xylene), and is generally preferred over D.P.X. Other recommended synthetic mounting
(Refractive Index 1.544) media include Permount (made by Fisher Scientific), H.S.R. (Harleco Synthetic Resin), and
Clearmount (Gurr).

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Mountants for Immunochemical Staining

The choice of mounting medium following immunochemical staining is largely dictated by the label (and in the case
of enzymatic labels, the chromogen) used to visualize the antigen. Aqueous mounting medium is generally suitable for all
enzymatic label/chromogen combinations and fluorescent labels. Specimens mounted in such media are mounted straight
from the aqueous phase (with no dehydration or clearing).

Aqueous mounting media for phycobiliprotein fluorescent labels (phycoerythrin, phycocyanin) must not contain
glycerol as this quenches the staining intensity. Similarly, exposure to excitation light of most fluorescent labels results in
diminished staining, a process known as photo bleaching. Apathy’s medium (Refractive Index 1.52) is the most useful
aqueous mountant for fluorescent microscopy, being virtually non-fluorescent. Polyvinyl alcohol (Refractive Index 1.5) is an
alternative for glycerine jelly, commonly used for fluorescent labels with paraphenylene-diamine as antifading agent. Ready-
touse anti-fading kits are also commercially available.

1. Cover slipping The stained section on the slide must be covered with a thin piece plastic or glass to protect
the tissue from being scratched, to provide better optical quality for viewing under the
microscope, and to preserve the tissue section for years to come. The stained slide must
go through the reverse process that it went through from paraffin section to water. The
stained slide is taken through a series of alcohol solutions to remove the water, then
through clearing agents to a point at which a permanent resinous substance beneath the
glass coverslip, or a plastic film, can be placed over the section.

Bubbles under the coverslip may form when the mounting media is too thin, and as it dries
air is sucked in under the coverslip. Contamination of clearing agents or cover slipping
media may also produce a bubbled appearance under the microscope

2. Ringing Ringing is the process of sealing the margins of the cover-slip to prevent the escape of
fluid or semi-fluid mounts and evaporation of mountant, to fix the coverslip in place, and to
prevent sticking of the slides upon storage. The term “ringing” originated because round
coverslips were initially used and the coating applied in the form of a circle or “ring.”

A liquid preparation sealed well with nail polish could last some months. Paraffin wax may
be applied with a ringing iron and is satisfactory as a temporary ringing agent. The ringing
media used may be Kronig cement made up of two parts paraffin wax mixed with 4-9 parts
powdered colophonium resin, heated and filtered. Also available are cellulose adhesives
such as Durofix.

3. Broken Slides Mounting a broken slide on to another clean xylene-moist slide with a drop of mounting
media (Clarite or Permount) may be sufficient for immediate examination while a new
section is being cut and stained. If an important slide is broken and replacement is not
available, the section (if still intact) may be transferred to another slide.

The coverslip can be removed by soaking in xylene, and placing the broken slide in the
incubator at 37°C until all the mountant has been removed. The whole slide is then covered
with a mixture of 6 parts butyl acetate and 1 part durofix and left in the incubator for 30
minutes until the mixture hardens into a film. Using a sharp scalpel blade, the hardened
film is cut around the section, and the slide is placed in cold water until the film and section
float off. The film containing the section is mounted on a clean slide, placed in the 37°C
incubator until dry, washed gently with butyl acetate, then washed well with xylene, and
mounted in Clarite or Permount.

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